Methods of identifying senp1 inhibitors

ABSTRACT

Provided herein are methods of detecting binding of an SENP1 polypeptide to a compound and methods for screening for inhibitors of SENP1. Further provided are aqueous compositions comprising SENP1 polypeptides and NMR apparatuses comprising the compositions for NMR analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Applications 61/809,208, filed Apr. 5, 2013, and 61/813,832, filed Apr. 19, 2013, each of which is incorporated herein by reference in its entirety and for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under NIH Grant Nos. R01GM074748, R01GM086171 and R01GM102538. The government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 95058-905089_ST25.TXT, created on Apr. 7, 2014, 22,240 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

BACKGROUND

Post-translational modifications with the small ubiquitin-like modifiers (SUMO) are initiated and removed by the activities of SUMO-specific proteases (SENPs). Unlike ubiquitylation, which has one modifier (i.e., ubiquitin) and one dominant role, namely protein degradation, SUMOylation involves three modifiers (SUMO-1, -2, and -3) and affects diverse cellular functions. There are six SENPs, organized into three families based on sequence similarity: SENP1 and 2 that catalyze maturation of SUMO precursors and removal of SUMO-1 and SUMO-2/3 conjugates; SENP3 and 5 that preferentially remove SUMO-2/3 conjugates; and SENP6 and 7 that appear to be mainly involved in editing poly-SUMO-2/3 chains. Recently, another de-SUMOylase has been discovered that does not share sequence similarity with the SENPs.

SENP inhibitors with cellular activity would be advantageous for elucidating the role of SUMOylation in cellular regulation and for validating SENPs as therapeutic targets. SENP1 and SENP3 are also potential targets for developing new therapeutic agents for cancer. They regulate the stability of hypoxia-inducible factor 1α (HIF1α), which is a key player in the formation of new blood vessels to support tumor growth. SENP1 is also highly expressed in human prostate cancer specimens and regulates androgen receptor (AR) activities. Androgen induces rapid and dynamic conjugation of SUMO-1 to AR, while SENP1 promotes AR-dependent transcription by cleaving SUMO-1-modified AR. SENP1 overexpression induces transformation of normal prostate gland tissue and facilitates the onset of high-grade prostatic intraepithelial neoplasia. Therefore, at least some members of the SENPs are potential targets for developing new cancer therapies.

SUMMARY

Provided herein are methods of detecting binding of an SENP1 polypeptide to a compound and methods for screening for inhibitors of SENP1. Further provided are aqueous compositions comprising SENP1 polypeptides and NMR apparatuses comprising the compositions for NMR analysis.

The details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a picture of a representative Coomassie-stained gel showing cleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the presence of increasing concentrations of SPI-01. YSE, fusion SUMO (S) precursors flanked by YFP (Y) and ECFP (E) at the N- and C-termini, respectively.

FIG. 2 is a picture of representative Coomassie-stained gel showing cleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the presence of increasing concentrations of SPI-07. YSE, fusion SUMO (S) precursors flanked by YFP (Y) and ECFP (E) at the N- and C-termini, respectively.

FIG. 3 is a graph showing the effects of the panel of inhibitors shown in Table 1 at inhibiting SENP1, 2 and 7. In 96-well plates, SENPs (50-200 nM) were pre-treated with increasing concentrations of each compound, after which DUB-Glo (40 μM final concentration; Promega, Madison, Wis.) was added as substrate. Experiments were performed in triplicate. The amount of cleaved product is proportional to the relative light unit (RLU), which is bioluminescence produced by luciferase catalyzed reaction of luciferin that was produced by SENP cleavage of DUB-Glo.

FIG. 4 is a picture of a gel showing accumulation of SUMO-2/3-modified proteins in HeLa cells upon treatment with increasing doses of SPI-01.

FIG. 5 is a picture of a gel showing retention of SUMOylated proteins during recovery of HeLa cells from heat shock in the presence of 60 μM SPI-01 and SPI-02.

FIG. 6 is a graph showing superimposition of a section of the 2D ¹H-¹⁵N-heteronuclear single quantum coherence (HSQC) spectra of the catalytically inactive C603S mutant of human SENP1 in the absence (black cross-peaks) and presence of SPI-01 (grey cross-peaks) at 25° C. Perturbed representative cross-peaks at or near the catalytic site of SENP1 are labeled.

FIG. 7 is a graph showing the superimposition of a section of the 2D ¹H-¹⁵N-HSQC spectra of SUMO-1 precursor showing labeled peaks of the C-terminal residues when free (black) and bound to SENP1-C603S (dark grey) or both SENP1-C603S and SPI-01 (light grey) at 35° C.

FIG. 8 is a picture showing all SPI-01 perturbed residues on SENP1 (PDB ID: 2IY1) labeled and colored in dark grey on the surface representation of SENP1 in complex with SUMO-1 precursor. Perturbed residues that are located in the vicinity of the catalytic center of SENP1 or the C-terminus of precursor SUMO-1 are labeled in black and grey, respectively.

FIGS. 9A and 9B are graphs showing enzyme kinetic measurements for SPI-01 indicating a non-competitive mode of inhibition. The data were fit to obtain the indicated kinetic parameters (α, K_(i) and K_(m)) using Graphpad Prism. Lineweaver-Burk plot analysis of the data also confirmed non-competitive inhibition.

DETAILED DESCRIPTION

SENP1 is a target for developing new therapeutic agents for cancer. It regulates the stability of hypoxia-inducible factor 1α (HIF1α), which is a key player in the formation of new blood vesicles to support tumor growth. SENP1 is also highly expressed in human prostate cancer specimens and regulates androgen receptor (AR) activities. SENP1 is also a target for developing anti-viral therapeutic agents for infection of viruses including, but not limited to influenza, cytomegalovirus, herpes virus, white spot syndrome virus, Epstein-Barr virus, adenovirus and HIV-1, because of the role of SUMOylation in their replication. As described in the examples below, small molecule inhibitors of SENP1 were searched for using in-silico screening in conjunction with biochemical assays. However, the data provided evidence for substrate-assisted inhibitor binding. Thus, using artificial substrates for compound screening may be misleading, as the inhibitory effects could be significantly different from using the physiological substrates. Therefore, embodiments are provided including methods and inhibitors of SENP1 that confer the non-competitive inhibitory mechanism, as shown by nuclear magnetic resonance (NMR).

For specific SENP proteins described herein (e.g., SENP1), the named protein includes any of the protein's naturally occurring forms, or variants that maintain the protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the SENP1 protein is the protein as identified by its NCBI sequence reference. In other embodiments, the SENP1 protein is the protein as identified by its NCBI sequence reference or functional fragment thereof.

The term “SENP1” as provided herein includes any of the Sentrin-specific protease 1 (SENP1) naturally occurring forms, homologs, isoforms or variants that maintain the protease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In embodiments, the SENP1 protein is the protein as identified by the NCBI sequence reference GI:390131988 or functional fragment thereof. In embodiments, the SENP1 protein is the protein as identified by the UniProt sequence reference Q9P0U3 or functional fragment thereof. In embodiments, the SENP1 protein includes the sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. In embodiments, the SENP1 protein is encoded by a nucleic acid sequences corresponding to Gene ID: 29843.

As described herein, nuclear magnetic resonance (NMR) approaches have advantages over other methods previously employed on SENP1 in identifying molecules or compounds for further development. Specifically, the methods herein provide for discovery or identification of compounds or inhibitors that selectively bind SENP1 and not other SENPs. The methods also provide for identification of compounds or inhibitors that selectively bind SENP1-physiological substrate complexes and not SENP-artificial substrate complexes. Further advantages include sensitivity to binding affinities of a wide range and, thus, allowing for identification of compounds with physicochemical properties that are amenable for a greater scope for development of leads with superior ADME (absorption, distribution, metabolism, and excretion) attributes. Optionally, the test compounds are Rule-of-three (Ro3) (MW≦300, H-bond donors/acceptors ≦3, c Log P≦3, rotatable bonds ≦3) compliant (Congreve et al., Drug Discov. Today 8(19):876-7 (2003); and Erlanson, Top Curr. Chem. 317:1-32 (2011)).

Nuclear magnetic resonance (NMR) studies magnetic nuclei and provide atomic resolution information on the structures of large or small molecules and their complexes. The elementary particles, neutrons and protons, composing an atomic nucleus, have the intrinsic quantum mechanical property of spin. The overall spin of the nucleus is determined by the spin quantum number I. If the number of both the protons and neutrons in a given isotope are even, then I=0. In other cases, however, the overall spin is non-zero. A non-zero spin is associated with a non-zero magnetic moment. It is this magnetic moment that is exploited in NMR. For example, nuclei that have a spin of one-half, like Hydrogen nuclei (¹H), a single proton, have two possible spin states (also referred to as up and down, respectively). The energies of these states are the same. Hence the populations of the two states (i.e. number of atoms in the two states) will be approximately equal at thermal equilibrium. If a nucleus is placed in a magnetic field, however, the interaction between the nuclear magnetic moment and the external magnetic field means the two states no longer have the same energy. The NMR frequency (f) is shifted by the shielding effect of the surrounding electrons. In general, this electronic shielding reduces the magnetic field at the nucleus (which is what determines the NMR frequency). As a result, the energy gap is reduced, and the frequency required to achieve resonance is also reduced. This shift of the NMR frequency due to the chemical environment is called the chemical shift, and it explains why NMR is a direct probe of chemical structure. The chemical shift in absolute terms is defined by the frequency of the resonance expressed with reference to a standard which is defined to be at 0. The scale is made manageable by expressing it in parts per million (ppm) of the standard frequency. Thus, in general, NMR spectral data are reported as chemical shift and are reported in ppm relative to either an internal standard or other baseline. A more detailed discussion of nuclear magnetic resonance may be found in, for example, C. P. Slichter, Principles of Magnetic Resonance, 3rd ed., Springer-Verlag, Berlin, pp. 1-63 (1990); J. D. Roberts, Nuclear Magnetic Resonance, Mc-Graw-Hill, N.Y., pp. 1-19 (1959); Cohen-Tannoudji et al., Quantum Mechanics, Vol. 1, New York, N.Y.: Wiley (1977); WO 2009/027973; WO 2009/029880; WO 2009/029896; Hajduk et al., “High-throughput nuclear magnetic resonance-based screening,” J. Med. Chem. 42:2315-2317 (1999); and Cavanagh et al., Protein NMR Spectroscopy: Principles and Practice Academic Press: San Diego (1996), which are incorporated by reference herein in their entireties.

A variety of NMR approaches have been developed to accelerate NMR data acquisition (Atreya et al., Methods Enzymol., 394:78-108 (2005)). For example, in the field of biological NMR spectroscopy (Cavanagh et al., Protein NMR Spectroscopy, Academic Press: San Diego (2007)) stable isotope (¹³C/¹⁵N) labeled biological macromolecules are now studied. The isotope labeling enables one to efficiently record three-dimensional (3D) or four-dimensional (4D) ¹³C/¹⁵N-resolved spectra. The most commonly used biological NMR methods are multi-dimensional and heteronuclear-edited NMR methods. See, for example, Tjandra and Bax, “Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium,” Science 1997 278(5340):1111-4 (1997). Erratum in: Science 278(5344):1697 (1997); Clore and Gronenborn, “NMR structure determination of proteins and protein complexes larger than 20 kDa,” Curr Opin Chem. Biol. October; 2(5):564-70 (1998); Mittermaier and Kay, “Observing biological dynamics at atomic resolution using NMR,” Trends Biochem Sci. 34(12):601-11 (2009); and Wüthrich, Kurt, NMR of Proteins and Nucleic Acids, John Wiley, New York, N.Y. (1986). NMR techniques further include, but are not limited to, (i) Reduced-dimensionality (RD) NMR (Szyperski et al., Proc. Natl. Acad. Sci. U.S.A., 99:8009-8014 (2002)); (ii) G-matrix FT (GFT) projection NMR (Atreya et al., J. Am. Chem. Soc., 127:4554-4555 (2005); Eletsky et al., J. Am. Chem. Soc., 127:14578-14579 (2005); Yang et al., J. Am. Chem. Soc., 127:9085-9099 (2005); Szyperski et al., Magn. Reson. Chem., 44:51-60 (2006); Atreya et al., J. Am. Chem. Soc., 129:680-692 (2007); Kupce et al., J. Am. Chem. Soc., 126:6429-40 (2004); Hiller et al., Proc. Natl. Acad. Sci. U.S.A., 102:10876-10881 (2005); and Eghbalnia et al., J. Am. Chem. Soc., 127: 12528-12536 (2005)); and (iii) Covariance NMR spectroscopy (Bruschweiler, J. Chem. Phys., 121:409-414 (2004); Zhang et al., J. Am. Chem. Soc., 126:13180-13181 (2004); and Chen et al., J. Am. Chem. Soc., 128:15564-15565 (2006)). These publications are incorporated by reference herein in their entireties.

Thus, as used herein, the term nuclear magnetic resonance (NMR) encompasses a variety of methods including but not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), transverse relaxation optimized spectroscopy (TROSY-NMR) and combinations thereof. For more description on TROSY-NMR see Pervushin, et al., “Attenuated T₂ relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution” PNAS 94:12366-71 (1997), which is incorporated by reference herein in its entirety.

As used herein, the term “chemical shift,” in nuclear magnetic resonance (NMR) spectroscopy, refers to the resonant frequency of a nucleus relative to a standard or baseline. Some atomic nuclei possess a magnetic moment (nuclear spin), which gives rise to different energy levels and resonance frequencies in a magnetic field. The electron distribution of the same type of nucleus (e.g. ¹H, ¹³C, ¹⁵N) usually varies according to the local geometry and with it the local magnetic field at each nucleus. This is reflected in the spin energy levels (and resonance frequencies). The variation of nuclear magnetic resonance frequencies of the same kind of nucleus, due to variations in the electron distribution, is called the chemical shift. The size of the chemical shift is typically given with respect to a reference frequency or reference sample usually a molecule with a barely distorted electron distribution. Typically, a ¹H-¹⁵N HSQC spectrum is used to obtain chemical shift values. However, as provided in the methods herein, any NMR analysis method can be used.

As used herein, the term “chemical shift of an amino acid” includes the chemical shift of an element within the amino acid, e.g., H, C or N. As used herein, the term “element” refers to an atom distinguished by its atomic number, which is the number of protons in its nucleus. Exemplary elements include, but are not limited to, H (hydrogen), N (nitrogen) and C (carbon).

Exemplary chemical shift values for certain amino acids in the SENP1 polypeptide are shown in Table 3 and exemplary chemical shift values for certain amino acids in the SENP1 polypeptide when bound to SUMO are shown in Table 4. The sample conditions that correlate to the chemical shifts listed in Table 3 are 20 mM sodium phosphate, at pH 6.8 at 25° C. The sample conditions that correlate to the chemical shifts listed in Table 4 are 20 mM sodium phosphate and containing 150 mM NaCl, at pH 7, at 35° C. The values of the chemical shifts listed in Table 3 and Table 4 may vary by as much as 1 ppm for ¹H, and as much as 5 ppm for ¹⁵N and ¹³C due to differences in experimental conditions such as sample pH, temperature, addition of other components (e.g., salt), or amino acid substitutions in SENP1 and/or SUMO that may affect the function of SENP1 and/or SUMO. Thus, the chemical shifts listed in Tables 3 and 4 may vary from 1 ppm for ¹H and from 5 ppm for ¹⁵N and ¹³C.

Thus, the peaks or chemical shifts in Tables 3 and 4 can be used by those of skill in the art to determine whether a test compound binds SENP1 by correlating experimental peaks or chemical shifts to those provided in Tables 3 and 4. For example, the peaks or chemical shifts obtained by NMR in the presence of a test compound can be compared to the corresponding peaks or chemical shifts in Tables 3 or 4 to determine whether the test compound binds SENP1. Thus, the chemical shift for an amino acid of SENP1 in Table 3 or 4 can be compared to the corresponding chemical shift obtained for the same amino acid in SENP1 in the presence of a test compound. When performing such comparisons, one of skill in the art will account for variances known to affect chemical shift values due to changes in experimental conditions, e.g., pH, temperature, addition of other components (e.g., salt), or amino acid substitutions. In some embodiments, detection of a change of greater than 5 ppm in the chemical shift for ¹⁵N or ¹³C of an amino acid of SENP1 or greater than 1 ppm in the chemical shift for ¹H of an amino acid of SENP1 indicates non-correlation of peaks. Optionally, the change is as compared to the corresponding chemical shift value for ¹⁵N, ¹³C, or ¹H of an amino acid of SENP1 in Table 3 or Table 4.

As used herein, the binding of a compound to SENP1 may be selective. The terms “selectively binds,” “selectively binding,” or “specifically binding” refers to the compound binding SENP1 to the partial or complete exclusion of other agents or compounds. By binding is meant a detectable binding, for example, binding above the background of the assay method. Optionally, detectable binding is evidenced by comparing baseline to experimental values, e.g., by comparing baseline NMR data (e.g., chemical shift values or digital resolution spectra) to experimental NMR data (e.g., chemical shift values or digital resolution spectra). Thus, binding can be determined by detecting changes or perturbations in an NMR measurement or spectrum for one sample, e.g., a control sample, compared to another or second sample, e.g., a sample containing a test compound. Detectable changes or perturbations in NMR signals include changes in location (chemical shift). General NMR techniques for proteins, including multidimensional NMR experiments and determination of protein-ligand interactions can be found in David G. Reid (ed.), Protein NMR Techniques, Humana Press, Totowa N.J. (1997). By way of example, detection of a perturbation or change includes detection of a difference in the chemical shift of SENP1 or SENP1-SUMO complex in the presence of a compound as compared to the chemical shift in the absence of the compound. The perturbation or change (whether increased or decreased) can include significant differences in an NMR measurement or spectrum (e.g., chemical shift) and can be greater than the experimental error or greater than the error bar range. For example, a change of at least about 1.1 times of the digital resolution of a spectrum or chemical shift for one or more amino acid residues of SENP1 in the presence of a compound can indicate the compound binds SENP1. Thus, a change of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20 times or more of the digital resolution of an NMR measurement or spectrum, e.g., chemical shift, observed in the presence of a compound as compared to a control can indicate the compound binds SENP1.

The terms greater, higher, increases, elevates, or elevation refer to increases above a control. The terms low, lower, reduces, or reduction refer to any decrease below control levels. For example, control levels are levels prior to, or in the absence of, addition of a compound.

A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound, and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

“Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 10 to about 100, about 20 to about 75, about 30 to about 50 amino acids or nucleotides in length. Optionally, the identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 500 or 1000 or more amino acids in length. The present invention includes polypeptides that are substantially identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

An amino acid residue in a polypeptide “corresponds” to or “is corresponding to” a given residue when it occupies the same essential structural position within the polypeptide as the given residue. For example, a selected residue in a comparison polypeptide corresponds to position 603 in a polypeptide provided herein (e.g., a SENP1 polypeptide), when the selected residue occupies the same essential spatial or structural relationship to position 603 as assessed using applicable methods in the art. For example, a comparison polypeptide may be aligned for maximum sequence homology with the polypeptide provided herein and the position in the aligned comparison polypeptide that aligns with position 603 may be determined to correspond to it. Alternatively, instead of (or in addition to) a primary sequence alignment as described above, a three dimensional structural alignment can also be used, e.g., where the structure of the comparison polypeptide is aligned for maximum correspondence with a polypeptide provided herein and the overall structures compared. In this case, an amino acid that occupies the same essential position as position 603 in the structural model may be said to correspond.

“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles.

The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The “active-site” of a protein or polypeptide refers to a protein domain that is structurally, functionally, or both structurally and functionally, active. For example, the active-site of a protein can be a site that catalyzes an enzymatic reaction, i.e., a catalytically active site. An active site refers to a domain that includes amino acid residues involved in binding of a substrate for the purpose of facilitating the enzymatic reaction. Optionally, the term active site refers to a protein domain that binds to another agent, molecule or polypeptide. For example, the active sites of SENP1 include sites on SENP1 that bind to or interact with SUMO. A protein may have one or more active-sites.

The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength pH. The T_(m) is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_(m), 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley & Sons.

For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).

The term “pharmaceutically acceptable salts” or “pharmaceutically acceptable carrier” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present application contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present application contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for compositions of the present application.

A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a fluorescent label into a peptide specifically reactive with a target peptide (e.g., SENP1 polypeptide, SUMO protein or test compound). In embodiments, the label is a fluorescent label. Any method known in the art for conjugating a polypeptide to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

A “labeled protein or polypeptide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the labeled protein or polypeptide may be detected by detecting the presence of the label bound to the labeled protein or polypeptide.

Methods

Provided herein are methods of detecting binding of an SENP1 polypeptide to a compound. The method includes the steps of contacting an SENP1 polypeptide with a compound, allowing the compound to bind to the SENP1 polypeptide, thereby forming a SENP1-compound complex, and detecting the SENP1-compound complex using nuclear magnetic resonance, thereby detecting binding of the SENP1 polypeptide to the compound.

A “compound” as provided herein refers to a polypeptide, protein, amino acid, small molecule or chemical compound that is capable of binding a SENP1 polypeptide or fragment thereof. In embodiments, the compound binds a SENP1 protein of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. In embodiments, the compound is a modulator of SENP1 activity. In embodiments, the compound is an inhibitor of SENP1 activity. In embodiments, the compound is an activator of SENP1 activity. In embodiments, the compound is a small molecule. A small molecule as provided herein include, but are not limited to the compounds in Tables 1 and 2 and those described in WO 2012/064887, which is incorporated by reference herein in its entirety. As used herein, the term “small molecule” refers to an organic compound containing carbon. A small molecule is generally, but not necessarily, of low molecular weight, e.g., less than 1000 Daltons.

A “test compound” as provided herein refers to a compound useful for the screening methods provided herein. A test compound may be capable of binding a SENP1 polypeptide or fragment thereof as provided herein. In embodiments, the test compound binds a SENP1 polypeptide or fragment thereof. In embodiments, the binding of the test compound to the SENP1 polypeptide or fragment thereof is detected by nuclear magnetic resonance. In embodiments, the test compound does not bind a SENP1 polypeptide or fragment thereof.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a compound or protein-inhibitor interaction means negatively affecting (e.g., decreasing) the activity or function of the protein (e.g. decreasing gene transcription or translation) relative to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer). In embodiments, inhibition refers to a reduction in the activity of an enzymatic activity (e.g., SENP activity). In embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway (e.g. cell cycle). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating transcription, translation, signal transduction or enzymatic activity or the amount of a protein (e.g. a cellular protein or a viral protein). In embodiments, inhibition refers to inhibition of SENP1.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance that results in a detectably lower expression or activity level as compared to a control. The inhibited expression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control. An “inhibitor” is a siRNA, (e.g., shRNA, miRNA, snoRNA), compound or small molecule that inhibits cellular function (e.g., replication) e.g., by binding, partially or totally blocking stimulation, decrease, prevent, or delay activation, or inactivate, desensitize, or down-regulate signal transduction, gene expression or enzymatic activity necessary for protein activity Inhibition as provided herein may also include decreasing or blocking a protein activity (e.g., activity of SENP1).

The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.

Optionally, the compound is a small molecule. Optionally, the step of detecting includes detecting a perturbation in the presence of the compound relative to the absence of the compound. For example, binding of a compound to SENP1 is detected if a perturbation is detected in an NMR measurement or spectrum in the presence of the compound as compared to or relative to the absence of the compound. Optionally, the step of detecting includes determining a chemical shift for an amino acid in an active site of the SENP1 polypeptide. Binding is detected by a change in the chemical shift in the presence of the compound relative to the corresponding chemical shift in the absence of the compound. Optionally, the active site is a catalytically active site. Optionally, the active site is a site involved in SUMO binding, e.g., the active site is a site on SENP1 that binds to the SUMO protein. Thus, the step of detecting includes determining a chemical shift for an amino acid involved in binding of SENP1 polypeptide to SUMO. Optionally, the chemical shift is determined for one or more amino acids of SEQ ID NOs: 3, 4, 5, 6 or 7.

Optionally, the chemical shift is determined for one or more amino acid residues selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1.

In embodiments, the change is a change in the chemical shift of amino acid residue D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 or Q596 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue D550 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue H533 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue C603 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue W465 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue W534 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue L466 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue G531 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue C535 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue M552 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue G554 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue E469 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of amino acid residue Q596 of SEQ ID NO:1.

In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 or Q596 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to D550 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to H533 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to C603 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to W465 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to W534 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to L466 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to G531 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to C535 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to M552 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to G554 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to E469 of SEQ ID NO:1. In embodiments, the change is a change in the chemical shift of an amino acid residue corresponding to Q596 of SEQ ID NO:1.

In embodiments, the SENP1 polypeptide includes amino acid residue 603 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 603 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 550 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 550 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 533 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 533 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 465 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 465 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 534 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 534 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 466 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 466 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 531 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 531 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 535 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 535 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 552 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 552 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 554 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 554 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 469 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 469 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid residue 596 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an amino acid residue corresponding to amino acid residue 596 of SEQ ID NO:1.

Optionally, the chemical shift is determined for a mutation at amino acid residue 603 of SEQ ID NO:1. Optionally, the mutation is C6035. Optionally, the chemical shift is determined for one or more amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide or SENP1-compound complex is bound to a SUMO protein thereby forming a SENP1-SUMO complex or SENP1-SUMO-compound complex. Optionally, the SUMO protein is a truncated SUMO protein. Optionally, the compound does not interact with C603 of SEQ ID NO:1 of SENP1, e.g., the compound does not covalently modify C603 of SENP1. Thus, the provided methods optionally include detecting binding by producing an NMR spectra of the SENP-1 compound complex and identifying a change in the NMR spectra relative to the absence of the compound. Optionally, the change is a change in the chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7. Optionally, the change is a change in the chemical shift of an amino acid selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596. Optionally, the change is a change in the chemical shift of the amino acid 5603. Optionally, the change is a change in the chemical shift of an amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Also provided is a method of screening for compounds that bind SENP1 including the steps of providing a first sample comprising SENP1 or an SENP1-SUMO complex, determining an NMR spectra of the first sample, providing a second sample comprising an SENP1-compound complex or an SENP1-SUMO-compound complex, and determining an NMR spectra of the second sample. Detection of a change in the NMR spectra in the second sample as compared to the first sample indicates the compound binds SENP1.

Provided are methods of screening for an inhibitor of SENP1. The methods include contacting a composition comprising an SENP1 polypeptide with a test compound and detecting whether the test compound binds the SENP1 polypeptide or fragment thereof by nuclear magnetic resonance.

Optionally, the step of detecting includes detecting a perturbation in the presence of the compound relative to the absence of the compound. For example, the test compound binds or inhibits SENP1 if a perturbation is detected in an NMR measurement or spectrum in the presence of the compound as compared to or relative to the absence of the compound. Optionally, the step of detecting comprises determining a chemical shift for one or more amino acids in the active site of the SENP1 polypeptide. The chemical shift in the presence of the compound will be changed relative to the corresponding chemical shift in the absence of the test compound if the test compound binds to SENP1. Optionally, the active site is a catalytically active site. Optionally, the active site is a site involved in SUMO binding, e.g., the active site is a site on SENP1 that binds to the SUMO protein. Thus, the step of detecting includes determining a chemical shift for an amino acid involved in binding of SENP1 polypeptide to SUMO. Optionally, the chemical shift is determined for one or more amino acids of SEQ ID NOs: 3, 4, 5, 6 OR 7. Optionally, the chemical shift is determined for one or more amino acid residues selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. Optionally, the chemical shift is determined for a mutation at amino acid residue 603 of SEQ ID NO:1. Optionally, the mutation is C603S. Optionally, the chemical shift is determined for one or more amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex. Optionally, the SUMO protein is a truncated SUMO protein. Optionally, the composition comprising the SENP1 polypeptide or SENP1-SUMO complex is an aqueous solution. Optionally, the composition is at a pH from about 6.0 to about 7.5. Optionally, the pH is about 6.8. Optionally, the composition comprises a buffering agent, a reducing agent, a base or combinations thereof. Optionally, the composition comprises sodium phosphate, D₂O, sodium azide, dithiothreitol or combinations thereof. The sodium phosphate can be present at about 20 mM. Optionally, the compound to be tested is a small molecule. Optionally, the compound does not interact with C603 numbered relative to SEQ ID NO:1 of SENP1, e.g., the compound does not covalently modify C603 of SENP1. Optionally, in the provided methods, the SENP1 binds the compound forming an SENP1-compound complex and the detecting comprises producing an NMR spectra of the SENP1-compound complex and identifying a change in the NMR spectra relative to the absence of the compound. Optionally, the change is a change in the chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7. Optionally, the change is a change in the chemical shift of an amino acid selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596. Optionally, the change is a change in the chemical shift of the amino acid 5603. Optionally, the change is a change in the chemical shift of an amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the change is a change in the chemical shift of an amino acid in the active site of SENP1. Optionally, the active site is a catalytically active site or a site that binds to the SUMO protein.

Also provided are methods of identifying an SENP1 inhibitor that include combining an SENP1 polypeptide, a SUMO protein, and a test compound in a reaction vessel, allowing the SENP1 polypeptide, SUMO protein and test compound to form a SENP1-SUMO-compound complex, and detecting the SENP1-SUMO-compound complex thereby identifying the compound as a SENP1 inhibitor. A “reaction vessel” as provided herein refers to a vial, tube, flask, bottle, syringe or other container means, into which the SENP1 polypeptide, SUMO protein and test compound are combined to allow the formation of a SENP1-SUMO-compound complex.

Optionally, one or more of the SENP1 polypeptide, SUMO protein or test compound is labeled. Optionally, the label is a fluorescent label. Optionally, the test compound comprises a fluorescent label. Optionally, the SUMO is a truncated SUMO protein. Optionally, the SUMO comprises amino acid residues 1-92 of the SUMO protein. Optionally, the SUMO protein comprises SEQ ID NO:8 or SEQ ID NO:9. Optionally, the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1. Optionally, the mutation is C603S. Optionally, the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the test compound is a small molecule. In the provided methods, the detecting can be performed by a variety of methods known to those skilled in the art and described in the example below. See, e.g., Protein-Ligand Interactions, Vol. 1008, Methods in Molecular Biology, Humana Press, Inc., Clifton, N.J., Williams and Daviter, Eds. (2013). For example, a wide variety of assays for detecting binding can be used including labeled in vitro protein-ligand binding assays, cell based assays, immunoassays, and the like. Optionally, detecting can be performed using solution-phase binding assays, e.g., fluorescent polarization. Thus, binding can be detected by fluorescent polarization (Rossi et al., Nat. Protoc. 6(3):365-87 (2011)). Optionally, binding is detected by detecting a change in the thermal properties of SENP1, e.g., the thermal property can be the melting temperature of SENP1. In some embodiments, the detecting is performed using nuclear magnetic resonance. Optionally, the detecting comprises producing an NMR spectra of the SENP1-SUMO-compound complex and identifying a change in the NMR spectra relative to the absence of the test compound. Optionally, the change is a change in the chemical shift of an amino acid in an active site of the SENP1 polypeptide. The active site can be, for example, a catalytically active site or a site that binds to the SUMO protein. Optionally, the amino acid is an amino acid of SEQ ID NOs: 3, 4, 5, 6 OR 7. Optionally, the amino acid is selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596. Optionally, the amino acid is 5603. Optionally, the amino acid is amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

As used throughout, the term “SENP1 polypeptide” refers to full length SENP1 and fragments thereof. The sequence and structure of the SENP1 polypeptide is known. (See above and Protein Data Bank (PDB) accession codes 2IYC and 2IY1; Shen et al., Nat. Struct. Mol. Biol. 13(12):1069-1077 (2006); and Xu et al., Biochem. J. 398(3):345-52 (2006)). Optionally, the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1. Optionally, the mutation is C6035. Optionally, the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Optionally, in the provided methods, SENP1 is bound to SUMO or a fragment thereof, e.g., a truncated SUMO protein. Thus, optionally, the SENP1 is bound to a SUMO protein thereby forming a SENP1-SUMO complex. Optionally, the SUMO protein is a truncated SUMO protein. Optionally, the SUMO protein is SEQ ID NO:8 or 9. As used herein, the term “truncated SUMO protein” refers to a SUMO protein or polypeptide that has been manipulated to remove at least one amino acid residue relative to wild-type SUMO, e.g., a SUMO protein or polypeptide that occurs in nature. Exemplary wild-type SUMO proteins include, but are not limited to, SEQ ID NO:9 and those found at GenBank Accession Nos. AAC50996.1, NP_(—)008868.3, NP_(—)001005849.1, P55854.2, and NP_(—)008867.2. Truncated SUMO proteins include, but are not limited to, SEQ ID NO:8. As used herein, the term “SUMO” refers to SUMO1, SUMO2, or SUMO3 or fragments thereof or complexes thereof, e.g., SUMO2/3. The nucleic acid and amino acid sequences for SUMO are known. See, for example, Hay, Mol. Cell. 18(1):1-12 (2005); and Yeh, et al., J. Biol. Chem., 284(13):8223-7 (2009). For example, nucleic acid and amino acid sequences for SUMO-1 can be found at GenBank Accession Nos. U67122.1 and AAC50996.1. Nucleic acid and amino acid sequences for SUMO-2 can be found at GenBank Accession Nos. NM_(—)006937.3, NM_(—)001005849.1, NP_(—)008868.3 and NP_(—)001005849.1. Nucleic acid and amino acid sequences for SUMO-3 can be found at GenBank Accession Nos. NM_(—)006936.2, P55854.2, and NP_(—)008867.2. Optionally, the SENP1 is bound to SUMO1 to form an SENP1-SUMO1 complex.

The provided SENP1 polypeptides and/or SUMO polypeptides and fragments thereof may contain one or more modifications, e.g., a conservative modification. As used herein, the term “modification” refers to a modification in a nucleic acid sequence of a gene or an amino acid sequence. Modifications include, but are not limited to, insertions, substitutions and deletions. Amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional modifications. Insertions include amino and/or terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place.

Modifications are generated using any number of methods known in the art. For example, site directed mutagenesis can be used to modify a nucleic acid sequence. One of the most common methods of site-directed mutagenesis is oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, an oligonucleotide encoding the desired change(s) in sequence is annealed to one strand of the DNA of interest and serves as a primer for initiation of DNA synthesis. In this manner, the oligonucleotide containing the sequence change is incorporated into the newly synthesized strand. See, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA, 82:488; Kunkel et al., 1987, Meth. Enzymol., 154:367; Lewis & Thompson, 1990, Nucl. Acids Res., 18:3439; Bohnsack, 1996, Meth. Mol. Biol., 57:1; Deng & Nickoloff, 1992, Anal. Biochem., 200:81; and Shimada, 1996, Meth. Mol. Biol., 57:157. Other methods are routinely used in the art to introduce a modification into a sequence. For example, modified nucleic acids are generated using PCR or chemical synthesis, or polypeptides having the desired change in amino acid sequence can be chemically synthesized. See, for example, Bang & Kent, 2005, Proc. Natl. Acad. Sci. USA, 102:5014-9 and references therein.

Also provided herein are nucleic acids encoding the polypeptides described throughout. It is understood that the nucleic acids that can encode those peptide, polypeptide, or protein sequences, variants and fragments thereof are also disclosed. This would include all degenerate sequences related to a specific polypeptide sequence, i.e. all nucleic acids having a sequence that encodes one particular polypeptide sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the polypeptide sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed polypeptide sequence.

Provided herein are compounds to be tested for their ability to bind and/or inhibit SENP1. As used herein, an inhibitor refers to an agent or compound that inhibits SENP1 directly or indirectly. For example, an inhibitor of SENP1 can inhibit the expression or activity of SENP1. Compounds to be tested in the provided methods include, but are not limited to, small molecules, peptides, nucleic acids and antibodies. Optionally, the compound to be tested is a small molecule. Optionally, the small molecule is an inhibitor of SENP1. Small molecule inhibitors of SENP1 include, but are not limited to the compounds in Tables 1 and 2 and those described in WO 2012/064887, which is incorporated by reference herein in its entirety. As used herein, the term “small molecule” refers to an organic compound containing carbon. A small molecule is generally, but not necessarily, of low molecular weight, e.g., less than 1000 Daltons.

Once a compound has been identified as binding to SENP1 and/or inhibiting SENP1, the compound can be further tested for its binding and/or inhibitory abilities using a variety of known methods including the methods described in the example below. Various assays for determining levels and activities of protein are available, such as amplification/expression methods, immunohistochemistry methods, FISH and shed antigen assays, southern blotting, or PCR techniques. Moreover, the protein expression or amplification may be evaluated using in vivo diagnostic assays.

Compositions and Apparatuses for NMR Analysis

Provided herein are compositions comprising a SENP1 polypeptide and NMR apparatuses comprising the compositions for NMR analysis. Optionally, the composition is an aqueous solution. Optionally, the aqueous solution comprises an SENP1 polypeptide at a pH from about 6.0 to about 7.5. For example, the pH can be about 6.8. The provided compositions or aqueous solutions can further include, for example, buffering agents, reducing agents, solvents, bases and combinations thereof. Buffering agents include, but are not limited to, phosphate or citrate buffers. Reducing agents include but are not limited to, dithiothreitol, and sodium borohydride. Bases include, but are not limited to, metal oxides and salts of carbanions, amides and hydrides. Solvents include, but are not limited to, dimethyl sulfoxide (DMSO) Optionally, the compositions can include sodium phosphate, DMSO, D₂O, sodium azide, dithiothreitol or combinations thereof. By way of example, the sodium phosphate can be present at about 20 mM. Optionally, the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex. Optionally, the SENP1 polypeptide is bound to a compound thereby forming a SENP1-compound complex. Optionally, the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO-compound complex. Optionally, the SUMO protein is a truncated SUMO protein. Optionally, the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 numbered relative to SEQ ID NO:1.

An NMR apparatus comprising an NMR sample container for NMR analysis, said NMR sample container comprising the aqueous composition or solution is also provided. NMR apparatuses are known and can be obtained from commercially available sources. Makers of NMR equipment include, but are not limited to, Bruker (Germany), Oxford Instruments (United Kingdom), General Electric (Fairfield, Conn.), Philips (Amsterdam, Netherlands), Siemens AG (Munich, Germany) and Agilent Technologies, Inc. (Santa Clara, Calif.).

Compositions

Provided herein are compositions including the inhibitors identified by the screening and binding methods provided herein. The compositions are, optionally, suitable for formulation and administration in vitro or in vivo. Optionally, the compositions comprise one or more of the provided agents and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21^(st) Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

The inhibitors are administered in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, intratumoral or inhalation routes. The administration may be local or systemic. The compositions can be administered via any of several routes of administration, including topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, or by installation via bronchoscopy. Thus, the compositions are administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

The compositions for administration will commonly comprise an agent as described herein (e.g. inhibitor of SENP1) dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.

Pharmaceutical formulations, particularly, of the modified viruses can be prepared by mixing the modified adenovirus (or one or more nucleic acids encoding the modified adenovirus) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers. Such formulations can be lyophilized formulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used. Acceptable carriers, excipients or stabilizers can be acetate, phosphate, citrate, and other organic acids; antioxidants (e.g., ascorbic acid) preservatives low molecular weight polypeptides; proteins, such as serum albumin or gelatin, or hydrophilic polymers such as polyvinylpyllolidone; and amino acids, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents; and ionic and non-ionic surfactants (e.g., polysorbate); salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants. The modified adenovirus (or one or more nucleic acids encoding the modified adenovirus) can be formulated at any appropriate concentration of infectious units.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the modified adenovirus suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

The inhibitors of SENP1 can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the provided methods, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically intratumorally, or intrathecally. Parenteral administration, intratumoral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced or infected by adenovirus or transfected with nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. Thus, the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.

Methods of Treatment

The provided inhibitors of SENP1 can be administered for therapeutic or prophylactic treatments or used in the laboratory. Thus, provided is a method of treating a proliferative disorder in a subject. The method includes administering the provided inhibitors of SENP1 or compositions to the subject. As described throughout, the pharmaceutical composition is administered in any number of ways including, but not limited to, intravenously, intravascularly, intrathecally, intramuscularly, subcutaneously, intraperitoneally, or orally. Optionally, the method further comprising administering to the subject one or more additional therapeutic agents. Optionally, the therapeutic agent is a chemotherapeutic agent.

As described throughout, the proliferative disorder can be cancer. Optionally, the proliferative disorder is selected from the group consisting of lung cancer, prostate cancer, colorectal cancer, breast cancer, thyroid cancer, renal cancer, liver cancer and leukemia. Optionally, the proliferative disorder is metastatic.

In therapeutic applications, compositions are administered to a subject suffering from a proliferative disease or disorder (e.g., cancer) in a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. A “patient” or “subject” includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications.

Optionally, the provided methods include administering to the subject one or more additional therapeutic agents. Thus, the provided methods can be combined with other cancer therapies, radiation therapy, hormone therapy, or chemotherapy. The combined administrations contemplates coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Combinations of agents or compositions can be administered either concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first followed by administration of the second agent). Thus, the term combination is used to refer to concomitant, simultaneous or sequential administration of two or more agents or compositions.

According to the methods provided herein, the subject is administered an effective amount of one or more of the agents provided herein. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response (e.g., killing of a cancer cell). The dosages, however, may be varied depending upon the requirements of the subject, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular subject. The dose administered to a subject, in the context of the provided methods should be sufficient to affect a beneficial therapeutic response in the patient over time. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Thus, effective amounts and schedules for administering the agent may be determined empirically by one skilled in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

Kits

Provided herein are kits for screening for compounds that bind or inhibit SENP1. The kits include a composition comprising an SENP1 polypeptide. Optionally, the composition is an aqueous solution. Optionally, the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 numbered relative to SEQ ID NO:1. Optionally, the aqueous composition comprising an SENP1 polypeptide is at a pH from about 6.0 to about 7.5. Optionally, the pH is about 6.8. Optionally, the compositions can further include, for example, buffering agents, reducing agents, bases and combinations thereof. Optionally, the compositions can include sodium phosphate, D₂O, sodium azide, dithiothreitol or combinations thereof. By way of example, the sodium phosphate can be present at about 20 mM. Optionally, the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex. Optionally, the SENP1 polypeptide or SENP1-SUMO complex is bound to a compound thereby forming a SENP1-compound complex or SENP1-SUMO-compound complex. Optionally, the SUMO protein is a truncated SUMO protein. In some embodiments, the kit comprises a container including a SENP1 polypeptide or SENP1-SUMO complex and, optionally, a second container including a SENP1-compound complex or SENP-SUMO-compound complex.

Further provided are kits including an inhibitor of SENP1. Optionally, the kit comprises one or more doses of an effective amount of a composition comprising a SENP1 inhibitor. Optionally, the composition is present in a container (e.g., vial or packet). Optionally, the kit further includes one or more additional therapeutic agents. Optionally, the therapeutic agent is a chemotherapeutic agent. Optionally, the kit comprises a means of administering the composition, such as, for example, a syringe, needle, tubing, catheter, patch, and the like. The kit may also comprise formulations and/or materials requiring sterilization and/or dilution prior to use.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the claims.

EXAMPLE Example 1 Identification and Characterization of a SENP Inhibitors

Enzymes called SENPs catalyze both the maturation of small ubiquitin-like modifier (SUMO) precursors and removal of SUMO modifications, which regulate essential cellular functions such as cell cycle progression, DNA damage response and intracellular trafficking. Some members, such as SENP1, are potential targets for developing cancer therapeutics. A search for small molecule inhibitors of SENPs was carried out using in-silico screening in conjunction with biochemical assays, and a new chemotype of small molecule inhibitors that non-covalently inhibit SENPs was identified. The inhibitors confer the non-competitive inhibitory mechanism, as shown by nuclear magnetic resonance (NMR) and quantitative enzyme kinetic analysis. The NMR data also provided evidence for substrate-assisted inhibitor binding, which indicates the need for caution in using artificial substrates for compound screening, as the inhibitory effects could be significantly different from using the physiological substrates.

In this study, it was purported to identify small molecule inhibitors of SENPs through in-silico screening in conjunction with enzyme kinetic, nuclear magnetic resonance (NMR) and cellular analyses. In silico screening was performed using Protein Data Bank (PDB) accession codes 2IYC and 2IY1 and by considering hydrogen bonding and hydrophobic interactions between the C-terminus of full-length SUMO-1 and SENP1. The GLIDE program (Friesner et al., Journal of Medicinal Chemistry 47:1739-1749 (2004)) was used to search the 250,000 compound library provided by the Developmental Therapeutics Program (DTP) of the National Cancer Institute, using the E-model scoring function of Cvdw, which is the sum of the van der Waals (Evdw) and electrostatic interaction energy terms (Eelec). Among the top hits, the dominant scaffolds were peptidomimetics and compounds that contained 2-fold symmetry. Forty compounds (100 μM) representing the dominant scaffolds were tested for their inhibitory effects on SENP1 and SENP2 for maturation of SUMO-1 and SUMO-2 precursors. The most potent compounds contained sulfonyl-benzene groups. Additional analogues of this group were obtained from DTP, and NSC5068, hereafter referred to as SPI-01 (SUMO protease inhibitor), was found to have the highest potency (Table 1). Available analogs of SPI-01 were obtained from DTP. Five compounds in this group (Table 1, SPI-06 to SPI-10) are “half” of the other compounds (Table 1, SPI-01 to SPI-05) and allowed the exploration of the activity requirements of the two-fold symmetric structure of SPI-01 to SPI-05. The inhibitory activity of these compounds on SENP1 and SENP2 was characterized using substrates that contained precursor SUMO-1 or SUMO-2 (S) flanked by yellow fluorescent protein (Y) at the N-terminus and enhanced cyan fluorescent protein (E) at the C-terminus (YSE) (Tatham and Hay, Methods Mol. Biol. 497:253-268 (2009)). Although the cleavage of the substrates can be detected by fluorescence resonance energy transfer (FRET), FRET could not be used because many of these compounds interfere with the FRET signal. Therefore, a gel-based assay was used to determine the inhibitory effects of all compounds on SENP1 and 2 (representative data shown in FIGS. 1 and 2), and the gel bands were quantified to determine the half maximum inhibitory concentrations (IC₅₀) (Table 1). The inhibitory effects of the compounds on the endopeptidase activities were not only enzyme-dependent, but also substrate-dependent. For SENP1-mediated cleavage of SUMO-1 precursor, only four of the compounds (SPI-01 to SPI-04) had half maximal inhibitory concentrations (IC₅₀) below 60 μM. The inhibitors were more potent for inhibiting SENP2 than SENP1 for cleavage of the SUMO-1 precursor. However, for cleavage of the SUMO-2 precursor, some compounds (i.e. SPI-01 and SPI-04) had similar potency for inhibiting SENP1 and SENP2, while others (i.e. SPI-07 and SPI-10) were more potent for inhibiting SENP1 than SENP2 or vice versa (i.e. SPI-06 and SPI-09) (Table 1). In addition to the differential effects on SENP1 and SENP2, SPI-01 had more than 10 fold less potency for inhibiting a de-ubiquitin enzyme isopeptidase T than inhibiting SENP2.

TABLE 1 Effect of inhibitors on inhibition of the maturation of SUMO precursors by SENP1 and SENP2. Compounds IC₅₀ (μM)-SUMO1 IC₅₀ (μM)-SUMO2 Structure Code† NCI ID‡ SENP1 SENP2 SENP1 SENP2

SPI-01 NSC5068 32.8 ± 1.82 1.42 ± 3.0 1.88 ± 2.2 1.1 ± 5.8

SPI-02 NSC16224 26.5 ± 1.86 3.42 ± 1.6 2.08 ± 2.0 2.70 ± 2.1

SPI-03 NSC8676 20.27 ± 2.47 5.17 ± 1.32 1.86 ± 2.3 3.0 ± 2.0

SPI-04 NSC34933 11.2 ± 1.7 1.6 ± 2.5 2.32 ± 2.6 2.15 ± 2.28

SPI-05 NSC5067 >60 19.7 ± 1.47 7.5 ± 1.6 4.6 ± 1.65

SPI-06 NSC70551 >60 3.62 ± 1.98 43.2 ± 2.2 10.7 ± 1.6

SPI-07 NSC58046 >60 >60 17.54 ± 4.9 28.06 ± 9.2

SPI-08 NSC22940 >60 4.1 ± 3.0 >60 41.06 ± 5.2

SPI-09 NSC42164 >60 23.36 ± 1.6 >60 26.96 ± 2.5

SPI-10 NSC45551 >60 34.21 ± 1.9 11.1 ± 3.7 36.44 ± 5.7 †Designation for our library of SUMO-protease inhibitors (SPI) ‡Designated by the National Cancer Institute

To determine whether other SENPs can be inhibited by this family of inhibitors, a distant SENP member, SENP7, was tested in parallel with SENP1 and SENP2 using a pentapeptide substrate that contained the Gly-Gly motif and luciferin, known as DUB-Glo (Promega, Madison, Wis.). Cleavage of luciferin by a SENP can be detected by a coupled bioluminescent assay using luciferase. The bioluminescent reporter was chosen instead of a fluorescent reporter to avoid interference by the compounds during detection. In addition, because SENP7 has different physiological substrates than SENP1 and SENP2 (Kolli et al., Biochemical Journal 430:335-344 (2010); and Shen et al., EMBO Rep. 13(4):339-46 (2012)), an advantage of DUB-Glo is that it can act as a common substrate for all SENPs, which enabled us to rule out substrate-specific effects. The dose-dependent inhibition of each SENP by the inhibitors was determined (FIG. 3), as was the IC₅₀ for inhibition of SENP1, 2 and 7 of all the compounds (Table 2). Most compounds had more similar inhibitory effects on SENP1 and SENP2 than on SENP7, consistent with their amino acid sequence similarities. In addition, the compounds were more potent for inhibiting SENP1 when DUB-Glo was used as a substrate than when SUMO-1 precursor was used (Tables 1 and 2). To rule out the possibility that these compounds used a promiscuous mechanism, the compounds were also tested in SUMOylation and ubiquitination reactions, which also depend on enzymes containing catalytic Cys residues. The compounds were noninhibitory in these assays. Furthermore, comparison of the DUB-Glo and the SUMO maturation assays revealed that the effect of SENP inhibitors could be highly substrate-specific.

TABLE 2 Inhibitory effect on SENP enzymatic activity using a bioluminescent peptide substrate SENP1 SENP2 SENP7 Structure Code† NCI ID‡ IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)

SPI-01 NSC5068  5.9 ± 1.4  2.9 ± 1.6 3.5 ± 1.5

SPI-02 NSC 16224  2.1 ± 1.9  2.0 ± 2.0 2.7 ± 1.8

SPI-03 NSC 8676  3.8 ± 1.5  2.4 ± 1.8 4.8 ± 1.4

SPI-04 NSC 34933  2.4 ± 1.8  2.3 ± 1.8 3.4 ± 1.5

SPI-05 NSC 5067 13.3 ± 1.3  8.5 ± 1.3 4.6 ± 1.5

SPI-06 NSC 70551  3.9 ± 1.4  3.7 ± 1.4 4.7 ± 1.7

SPI-07 NSC 58046 >>60 >>60 1.9 ± 2.2

SPI-08 NSC 22940 22.2 ± 1.5 17.2 ± 1.5  2.8 ± 1.6

SPI-09 NSC 42164 >60  6.8 ± 1.3 1.9 ± 2.1

SPI-10 NSC 45551  2.4 ± 1.8  2.5 ± 1.7 2.0 ± 2.0 †Designation for our library of SUMO-protease inhibitors (SPI) ‡Designated by the National Cancer Institute

The abilities of representative inhibitors were then tested to inhibit SENP in cells. HeLa cells were treated with increasing concentrations of SPI-01 for 48 hours, after which SUMOylated proteins were detected in the cells by Western blots. SUMO-2/3 conjugates accumulated in cells and this accumulation correlated with inhibitor concentration, particularly at high molecular weights (FIG. 4). This result suggests that SPI-01 inhibits the isopeptidase activities of SENPs, particularly SENP6 and SENP7, which are required for SUMO chain editing. It was observed that less significant effects on the accumulation of SUMO-1 conjugates, possibly because most SENPs cleave SUMO-2/3-conjugates. It is known that heat shock triggers a dramatic increase in global SUMO-2/3 conjugations and that during recovery, the SUMOylated proteins are removed, at least in part, due to the deSUMOylation activity of SENP1 (Nefkens et al., J. Cell Sci. 116:513-524 (2003)). To further confirm that the inhibitors inhibited deSUMOylase activities, HeLa cells were treated with SPI-01 and SPI-02 for 2 hours at 37° C. Then, SPI-treated or untreated control HeLa cells was transferred to 42° C. for 30 minutes, followed by recovery for 4 hours at 37° C. before processing for detection of global SUMO-2/3 levels. The inhibitor-treated cells had considerably higher levels of SUMOylated proteins than did the corresponding controls that did not receive heat shock or the mock-treated cells after the recovery period (FIG. 5). Thus, the results of the heat-shock experiments further confirmed that the SPI compounds had inhibitory effects on SENPs in cells.

NMR chemical shift perturbation (CSP) analysis was used to investigate whether this family of inhibitors binds the enzyme or the enzyme-substrate complex. CSP experiments were conducted using a ¹⁵N-labeled C603S mutant of the human SENP1 catalytic domain (SENP1-C603S, for which NMR chemical shift assignments have been obtained and deposited in the Biological Magnetic Resonance Bank (BMRB) with accession number 19083). Although the SENP1-C603S mutant is catalytically inactive (Xu et al., Biochem. J. 398:345-52 (2006)), it retains binding activity for the precursor or mature SUMO paralogs or SUMOylated substrates (Shen et al., Nat. Struct. Mol. Biol. 13:1069-1077 (2006)). It was observed that SPI-01 caused modest backbone amide CSP for a subset of SENP1-C603S residues. Of note, specific CSPs were observed at the canonical cysteine-protease catalytic triad residues (D550, H533, and C603), the proposed dynamic channel of conserved W465 and W534, and at several other residues located at or adjacent to the SENP catalytic center (W465, L466, G531, H533, W534, C535, M552, G554 and Q596) with only one residue located distal to this surface (E469) (FIG. 2A). Interestingly, M552, G554, and Q596 are clustered at the SENP1 surface that contacts the C-terminal tail of SUMO-1. Supporting the importance of this surface in SENP catalytic activity, non-conservative point mutations of Q596 in SENP1 or the equivalent residue to SENP1 M552 in SENP2 (M497) perturb SUMO processing and deconjugation (Reverter and Lima, Nat. Struct. Mol. Biol. 13; 1060-8 (2006); and Shen et al., Biochem. J. 397:279-288 (2006)). Residue E469 is positioned toward the binding surface for the structured region of SUMO-1, and its CSP may be due to an alternative interaction with the compound or long-range effects. These results indicate that SPI-01 binds the surface adjacent to the catalytic center that contacts the C-terminal portion of the SUMO precursors. The residues that showed CSP are highly conserved between SENP1 and SENP2, suggesting that SPI-01 can interact with the equivalent surface on SENP2.

The binding of SPI-01 to the enzyme-substrate complex was investigated. CSP analysis was carried out on the 40 kDa complex of ¹⁵N-labeled full length precursor SUMO-1-GGHSTV (SUMO-1-FL) with unlabeled SENP1-C6035. An equimolar amount of SPI-01 was added to the 1:1 enzyme-substrate complex. The only observed CSP on the ¹⁵N-labeled precursor SUMO-1-FL was on the C-terminal residues S99 and V101 (FIGS. 7 and 8) (Song et al., PNAS 101:14373-8 (2004)). This result indicates that SPI-01 binds the enzyme-substrate complex at the interface between SENP and the C-terminal tails of precursor SUMO-FL. X-ray crystal structures showed that the C-terminal tail of precursor SUMO sits in and projects out of the catalytic tunnel of SENPs (Shen et al., Nat. Struct. Mol. Biol. 13:1069-77 (2006)). In the case of SENP1, the region that interacts with the projected C-terminus is predominantly acidic and favors the C-terminus of SUMO-1, which is polar and positively charged, over that of SUMO-2, whose C-terminus is mainly hydrophobic (Shen et al., Nat. Struct. Mol. Biol. 13:1069-77 (2006); and Shen et al., The Biochemical Journal 397:279-88 (2006)). In addition, the more hydrophobic C-terminus of SUMO-2 may favor binding of aromatic inhibitors. These properties may account for the more potent inhibition of processing of the SUMO-2 precursor (Table 1).

To further investigate the inhibitory mechanism, enzyme kinetic experiments were conducted using the pentapeptide substrate DUB-Glo (FIG. 9). The data was fit to a mixed inhibition mechanism, as described by the kinetic equation:

$v = \frac{V_{{ma}\; x}\lbrack S\rbrack}{\left( {1 + \frac{\lbrack I\rbrack}{{\alpha \; K_{i}}\;}} \right)\left\lbrack {\frac{K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)}{1 + \frac{\lbrack I\rbrack}{\alpha \; K_{i}}} + \lbrack S\rbrack} \right\rbrack}$

in which the value of “α” indicates the mechanism of inhibition (Segel, Enzyme Kinetics John Wiley & Sons (1993)). For both SENP1 and SENP2, the “α” values indicated that the inhibitory mechanism is mainly noncompetitive and suggests that the inhibitor binds to the enzyme and the enzyme-substrate complex to inhibit chemical conversion. This finding is consistent with the NMR binding analysis indicating that the inhibitor binds both the enzyme and the enzyme-substrate complex as discussed above.

In conclusion, this study has identified SENP inhibitors that do not covalently modify the catalytic Cys residue. This study has also provided the first mechanistic insights into how a small molecule inhibitor of SENPs that does not covalently modify the catalytic Cys can inhibit the enzymes. The substrate-assisted inhibitor binding indicates the need for caution in designing high throughput screening assays that use fluorogenic or chemiluminescent artificial substrates, as the results could be significantly different from using the physiological substrates. The substrate-dependent inhibitory effect suggests the possibility of designing SENP inhibitors that are tuned for substrate-specificity.

Materials and Methods

Protein Purification. The catalytic domains of SENP1, 2, and 7 were expressed as His-tagged protein in E. coli (DE3) and purified using nickel affinity chromatography (Namanja et al., The Journal of Biological Chemistry 287:3231-3240 (2012)). The pET11 expression plasmids for SENP1 and 2 contained a cDNA insert coding for the catalytic domain of human SENP1-WT (419-644) and SENP2-WT (364-589). The expression plasmid for the SENP1 active site point mutant C603S was generated using the QuikChange mutagenesis kit (Agilent Technologies, San Diego, Calif.). The expression plasmid for the catalytic domain of SENP7 has been described (Mikolajczyk et al., Journal of Biological Chemistry 282:26217-26224 (2007)).

SUMO Cleavage Assays. SUMO cleavage assays were performed by incubating SENPs with various concentrations of the inhibitor (0-60 μM) at room temperature for 10 min in assay buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM DTT). SENP concentrations were 32-50 nM when 50 μg/ml of the final substrate YFP-SUMO-ECFP (YSE) fusion protein was added. The mixture was incubated (37° C., 15 min), followed by SDS-PAGE and Coomassie staining for visualization. For cellular SENP inhibition experiments, HeLa cells cultured in DMEM plus 10% FBS, 100 units/ml penicillin, 100 mg/ml streptomycin, and 0.2 M glutamine were treated for 48 hours with SPI compounds. For heat shock experiment, HeLa cells were treated with SPI compounds or mock treated (2 h, 37° C.), after which cells were transferred to 42° C. for 30 min. After heat shock, the cells were allowed to recover (4-5 hours) before being harvested and lysed. Proteins were separated by SDS-PAGE and immunoblotted to determine global SUMO-2/3 levels.

DUB-Glo Assay. The luciferase substrate assay (DUB-Glo, Promega, Madison, Wis.) was performed according to the manufacturer's instructions. Briefly, SENPs (final concentration 50-100 nM) in Tris buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 10 mM DTT) were pre-incubated (10 min, room temperature) with increasing concentrations of inhibitor (0-60 μM final concentration) followed by addition of the luciferase substrate. Luciferase output was recorded 30 min after addition of the luciferase substrate. Values are the averages of experiments performed in triplicate.

NMR Experiments. Samples used for NMR titration or chemical shift perturbation analyses were ¹⁵N or ¹⁵N/¹³C-labeled; the titrant protein or SPI-01 was not labeled. The ¹⁵N/¹³C SUMO-1-FL sample was used to extend the backbone assignments of mature SUMO-1 to the HSTV tail by using 2D-¹⁵N-¹H-HSQC, 3D-HNCA, 3D-HNCOCA, and 3D-HNCACB. Additionally, comparison of ¹⁵N-¹H-HSQC between precursor and mature SUMO quickly identified the resonances of the HSTV tail. For SENP1 assignments, a full suite of triple-resonance NMR experiments were acquired on ¹⁵N/¹³C/²H or ¹⁵N/¹³C samples: HNCA, HNCOCA, HNCACB, HNCOCACB, HNCO, HNCACO, and NOESY-HSQC. All samples were dissolved in the NMR buffer: 20 mM sodium phosphate (pH 6.8), 10% D2O, 0.03% sodium azide and 10 mM d10-dithiothreitol. Purified perdeuterated SENP1 samples were unfolded and refolded into NMR buffer.

For titration of SENP1-C6035 with SPI-01, 270 μM ¹⁵N-labeled sample was titrated with the inhibitor that was prepared by diluting a 10 mM stock in 100% DMSO-d₆ to a concentration of 1.7 mM in the NMR buffer. The 2D ¹H-¹⁵N-HSQC spectra of SENP1 were recorded at each incremental addition of 5 μl of SPI-01 into 250 μl of SENP1. The chemical shift perturbation (CSP) analysis compared the spectra of SENP1 in the absence or the presence of equimolar SPI-01. A separate DMSO control titration was performed to account for DMSO-induced CSP. NMR resonance assignments for SUMO samples at 35° C. were transferred from those obtained at 25° C. by spectral acquisition at 2.5° C. incremental increases. All data were acquired on a 600 MHz Bruker Avance NMR spectrometer equipped with a TXI Cryoprobe.

TABLE 3 Free SENP1 NMR Chemical Shifts Values. Chemical Shift Ambiguity Index Value Definitions The values other than 1 are used for those atoms with different chemical shifts that cannot be assigned to stereospecific atoms or to specific residues or chains. Index Value Definition 1 Unique (including isolated methyl protons germinal atoms, and geminal methyl groups with identical chemical shifts (e.g. ILE HD11, HD12, HD13 protons) 2 Ambiguity of geminal atoms or geminal methyl proton groups (e.g. ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, or LEU HD11, HD12, HD13 and HD21, HD22, HD23 methyl protons) 3 Aromatic atoms on opposite sides of symmetrical rings (e.g. TYR HE1 and HE2 protons) 4 Intraresidue ambiguities (e.g. LYS HG and HD protons or TRP HZ2 and HZ3 protons) 5 Interresidue ambiguities (LYS 12 vs. LYS 27) 6 Intermolecular ambiguities (e.g. ASP 31 CA in monomer 1 and ASP 31 CA in monomer 2 of an asymmetrical homodimer, duplex DNA assignments, or other assignments that may apply to atoms in one or more molecule in the molecular assembly) 9 Ambiguous, specific ambiguity not defined Chemical Atom Residue Amino Atom Atom Iso- shift Unique- number number acid context type type (ppm)* ness 1 419 E CA C 13 56.635 1 2 419 E CB C 13 29.326 1 3 419 E CO C 13 175.803 1 4 419 E H H 1 8.056 1 5 419 E N N 15 120.257 1 6 420 F CA C 13 54.951 1 7 420 F CB C 13 37.882 1 8 420 F CO C 13 173.207 1 9 420 F H H 1 8.035 1 10 420 F N N 15 118.648 1 11 422 E CA C 13 56.498 1 12 422 E CB C 13 29.476 1 13 422 E CO C 13 176.111 1 14 422 E H H 1 8.637 1 15 422 E N N 15 124.065 1 16 423 I CA C 13 60.725 1 17 423 I CB C 13 35.427 1 18 423 I CO C 13 176.659 1 19 423 I H H 1 8.522 1 20 423 I N N 15 122.041 1 21 424 T CB C 13 70.292 1 22 424 T CO C 13 174.725 1 23 424 T H H 1 7.633 1 24 424 T N N 15 121.188 1 25 425 E CA C 13 59.696 1 26 425 E CB C 13 28.462 1 27 425 E H H 1 8.913 1 28 425 E N N 15 120.9 1 29 426 E CA C 13 59.444 1 30 426 E CO C 13 179.787 1 31 426 E H H 1 8.419 1 32 426 E N N 15 118.024 1 33 427 M CB C 13 33.371 1 34 427 M CO C 13 177.912 1 35 427 M H H 1 7.366 1 36 427 M N N 15 119.301 1 37 428 E CB C 13 28.41 1 38 428 E CO C 13 178.828 1 39 428 E H H 1 8.605 1 40 428 E N N 15 118.58 1 41 429 K CA C 13 59.488 1 42 429 K CB C 13 31.407 1 43 429 K CO C 13 178.978 1 44 429 K H H 1 7.858 1 45 429 K N N 15 118.101 1 46 430 E CB C 13 29.584 1 47 430 E CO C 13 179.111 1 48 430 E H H 1 7.356 1 49 430 E N N 15 119.087 1 50 431 I CA C 13 64.567 1 51 431 I CB C 13 38.123 1 52 431 I CO C 13 176.796 1 53 431 I H H 1 8.075 1 54 431 I N N 15 119.774 1 55 432 K CA C 13 59.195 1 56 432 K CB C 13 31.197 1 57 432 K CO C 13 180.185 1 58 432 K H H 1 8.32 1 59 432 K N N 15 116.686 1 60 433 N CA C 13 55.822 1 61 433 N CB C 13 37.95 1 62 433 N CO C 13 178.466 1 63 433 N H H 1 7.635 1 64 433 N N N 15 114.913 1 65 434 V CA C 13 64.189 1 66 434 V CB C 13 30.396 1 67 434 V CG1 C 13 22.475 1 68 434 V CG2 C 13 21.674 1 69 434 V CO C 13 176.412 1 70 434 V H H 1 7.548 1 71 434 V HG1 H 1 0.724 1 72 434 V HG2 H 1 0.725 1 73 434 V N N 15 114.74 1 74 435 F CA C 13 55.321 1 75 435 F CB C 13 37.701 1 76 435 F CO C 13 177.18 1 77 435 F H H 1 7.344 1 78 435 F N N 15 117.562 1 79 436 R CA C 13 56.384 1 80 436 R CB C 13 30.009 1 81 436 R CO C 13 176.099 1 82 436 R H H 1 7.225 1 83 436 R N N 15 118.73 1 84 437 N CA C 13 53.605 1 85 437 N CB C 13 38.193 1 86 437 N H H 1 8.252 1 87 437 N N N 15 119.685 1 88 438 G CA C 13 44.807 1 89 438 G CO C 13 172.725 1 90 438 G H H 1 8.08 1 91 438 G N N 15 109.481 1 92 439 N CA C 13 52.847 1 93 439 N CB C 13 37.175 1 94 439 N H H 1 8.737 1 95 439 N N N 15 120.443 1 96 440 Q CA C 13 58.04 1 97 440 Q CB C 13 28.495 1 98 440 Q CO C 13 175.605 1 99 440 Q H H 1 9.022 1 100 440 Q N N 15 125.59 1 101 441 D CA C 13 53.428 1 102 441 D CB C 13 40.409 1 103 441 D CO C 13 175.45 1 104 441 D H H 1 7.969 1 105 441 D N N 15 114.845 1 106 442 E CA C 13 56.501 1 107 442 E CB C 13 30.044 1 108 442 E CO C 13 175.905 1 109 442 E H H 1 7.143 1 110 442 E N N 15 121.501 1 111 443 V CA C 13 64.02 1 112 443 V CB C 13 31.155 1 113 443 V CG1 C 13 21.487 1 114 443 V CG2 C 13 21.844 1 115 443 V H H 1 8.65 1 116 443 V HG1 H 1 0.721 1 117 443 V HG2 H 1 0.882 1 118 443 V N N 15 127.026 1 119 444 L CA C 13 54.051 1 120 444 L CB C 13 43.293 1 121 444 L CD1 C 13 27.029 1 122 444 L CD2 C 13 21.807 1 123 444 L CO C 13 176.79 1 124 444 L H H 1 9.012 1 125 444 L HD1 H 1 0.59 1 126 444 L HD2 H 1 0.597 1 127 444 L N N 15 127.296 1 128 445 S CA C 13 57.432 1 129 445 S CB C 13 64.093 1 130 445 S CO C 13 172 1 131 445 S H H 1 7.412 1 132 445 S N N 15 111.726 1 133 446 E CA C 13 55.317 1 134 446 E CB C 13 31.965 1 135 446 E CO C 13 174.257 1 136 446 E H H 1 7.933 1 137 446 E N N 15 125.063 1 138 447 A CA C 13 51.875 1 139 447 A CB C 13 18.979 1 140 447 A CO C 13 176.087 1 141 447 A H H 1 8.286 1 142 447 A N N 15 124.213 1 143 448 F CA C 13 56.316 1 144 448 F CB C 13 36.029 1 145 448 F CO C 13 175.788 1 146 448 F H H 1 8.61 1 147 448 F N N 15 115.367 1 148 449 R CA C 13 57.675 1 149 449 R CB C 13 26.229 1 150 449 R CO C 13 175.491 1 151 449 R H H 1 8.484 1 152 449 R N N 15 110.844 1 153 450 L CA C 13 53.763 1 154 450 L CB C 13 44.035 1 155 450 L CD1 C 13 25.813 1 156 450 L CD2 C 13 22.437 1 157 450 L CO C 13 176.645 1 158 450 L H H 1 8.389 1 159 450 L HD1 H 1 0.89 1 160 450 L HD2 H 1 0.948 1 161 450 L N N 15 121.623 1 162 451 T CA C 13 60.834 1 163 451 T CB C 13 71.178 1 164 451 T CO C 13 173.407 1 165 451 T H H 1 8.315 1 166 451 T N N 15 113.296 1 167 452 I CA C 13 56.522 1 168 452 I CB C 13 36.082 1 169 452 I CO C 13 176.082 1 170 452 I H H 1 8.521 1 171 452 I N N 15 124.173 1 172 453 T CA C 13 59.709 1 173 453 T CB C 13 72.939 1 174 453 T H H 1 9.811 1 175 453 T N N 15 119.807 1 176 454 R CA C 13 60.137 1 177 454 R CB C 13 28.989 1 178 454 R CO C 13 177.392 1 179 454 R H H 1 8.211 1 180 454 R N N 15 122.061 1 181 455 K CA C 13 59.289 1 182 455 K CB C 13 31.114 1 183 455 K CO C 13 178.628 1 184 455 K H H 1 8.504 1 185 455 K N N 15 119.122 1 186 456 D CA C 13 57.369 1 187 456 D CB C 13 40.809 1 188 456 D H H 1 7.271 1 189 456 D N N 15 117.779 1 190 457 I CA C 13 62.392 1 191 457 I CB C 13 37.06 1 192 457 I H H 1 8.159 1 193 457 I N N 15 121.588 1 194 458 Q CA C 13 57.804 1 195 458 Q CB C 13 26.567 1 196 458 Q CO C 13 178.732 1 197 458 Q H H 1 7.923 1 198 458 Q N N 15 117.897 1 199 459 T CA C 13 65.051 1 200 459 T CB C 13 67.395 1 201 459 T H H 1 7.897 1 202 459 T N N 15 113.263 1 203 460 L CA C 13 54.923 1 204 460 L CB C 13 41.723 1 205 460 L CD1 C 13 25.968 1 206 460 L CD2 C 13 25.889 1 207 460 L CO C 13 179.644 1 208 460 L H H 1 7.253 1 209 460 L HD1 H 1 0.82 1 210 460 L HD2 H 1 0.925 1 211 460 L N N 15 115.083 1 212 461 N CA C 13 51.888 1 213 461 N CB C 13 37.194 1 214 461 N H H 1 7.421 1 215 461 N N N 15 119.845 1 216 462 H CA C 13 57.014 1 217 462 H CB C 13 28.992 1 218 462 H H H 1 7.773 1 219 462 H N N 15 119.821 1 220 465 W CA C 13 56.901 1 221 465 W CB C 13 27.801 1 222 465 W H H 1 8.319 1 223 465 W HE1 H 1 10.206 1 224 465 W N N 15 120.321 1 225 465 W NE1 N 15 130.435 1 226 466 L CA C 13 57.74 1 227 466 L CB C 13 41.916 1 228 466 L CD1 C 13 25.446 1 229 466 L CD2 C 13 23.298 1 230 466 L H H 1 7.644 1 231 466 L HD1 H 1 0.634 1 232 466 L HD2 H 1 0.563 1 233 466 L N N 15 125.508 1 234 467 N CA C 13 50.295 1 235 467 N CB C 13 39.556 1 236 467 N CO C 13 174.619 1 237 467 N H H 1 7.164 1 238 467 N N N 15 116.901 1 239 468 D CA C 13 57.481 1 240 468 D CB C 13 40.531 1 241 468 D H H 1 8.246 1 242 468 D N N 15 115.434 1 243 469 E CA C 13 60.578 1 244 469 E CB C 13 27.414 1 245 469 E CO C 13 179.948 1 246 469 E H H 1 8.991 1 247 469 E N N 15 119.089 1 248 470 I CA C 13 61.231 1 249 470 I CB C 13 34.744 1 250 470 I CO C 13 177.04 1 251 470 I H H 1 7.753 1 252 470 I N N 15 117.845 1 253 471 I CA C 13 64.903 1 254 471 I H H 1 6.974 1 255 471 I N N 15 117.941 1 256 472 N CA C 13 56.07 1 257 472 N CB C 13 37.624 1 258 472 N CO C 13 178.288 1 259 472 N H H 1 9.03 1 260 472 N N N 15 115.254 1 261 473 F CA C 13 62.584 1 262 473 F CB C 13 39.667 1 263 473 F CO C 13 177.436 1 264 473 F H H 1 8.304 1 265 473 F N N 15 123.63 1 266 474 Y CA C 13 62.784 1 267 474 Y CB C 13 38.335 1 268 474 Y CO C 13 178.151 1 269 474 Y H H 1 8.774 1 270 474 Y N N 15 120.467 1 271 475 M CA C 13 57.325 1 272 475 M CB C 13 31.041 1 273 475 M CO C 13 179.367 1 274 475 M H H 1 8.709 1 275 475 M N N 15 115.346 1 276 476 N CA C 13 56.292 1 277 476 N CB C 13 37.912 1 278 476 N CO C 13 177.604 1 279 476 N H H 1 7.371 1 280 476 N N N 15 117.074 1 281 477 M CA C 13 59.952 1 282 477 M CB C 13 31.504 1 283 477 M CO C 13 179.545 1 284 477 M H H 1 7.664 1 285 477 M N N 15 121.465 1 286 478 L CA C 13 57.471 1 287 478 L CB C 13 39.79 1 288 478 L CD1 C 13 27.34 1 289 478 L CD2 C 13 22.112 1 290 478 L CO C 13 180.857 1 291 478 L H H 1 7.767 1 292 478 L HD1 H 1 0.658 1 293 478 L HD2 H 1 0.411 1 294 478 L N N 15 119.925 1 295 479 M CA C 13 59.413 1 296 479 M CB C 13 32.433 1 297 479 M CO C 13 179.134 1 298 479 M H H 1 7.603 1 299 479 M N N 15 118.957 1 300 480 E CA C 13 59.225 1 301 480 E CB C 13 28.37 1 302 480 E H H 1 8.059 1 303 480 E N N 15 122.932 1 304 481 R CA C 13 58.251 1 305 481 R CB C 13 28.749 1 306 481 R CO C 13 176.421 1 307 481 R H H 1 7.917 1 308 481 R N N 15 120.662 1 309 482 S CA C 13 60.255 1 310 482 S CB C 13 63.102 1 311 482 S CO C 13 172.394 1 312 482 S H H 1 7.201 1 313 482 S N N 15 113.273 1 314 483 K CA C 13 56.793 1 315 483 K CB C 13 31.687 1 316 483 K CO C 13 178.176 1 317 483 K H H 1 6.968 1 318 483 K N N 15 118.755 1 319 484 E CB C 13 29.049 1 320 484 E CO C 13 176.653 1 321 484 E H H 1 8.114 1 322 484 E N N 15 121.011 1 323 485 K CA C 13 57.55 1 324 485 K CB C 13 31.154 1 325 485 K CO C 13 177.924 1 326 485 K H H 1 8.263 1 327 485 K N N 15 121.725 1 328 486 G CA C 13 44.731 1 329 486 G CO C 13 173.993 1 330 486 G H H 1 8.738 1 331 486 G N N 15 111.446 1 332 487 L CA C 13 52.224 1 333 487 L CB C 13 40.075 1 334 487 L CD1 C 13 25.797 1 335 487 L CD2 C 13 23.228 1 336 487 L CO C 13 174.966 1 337 487 L H H 1 7.357 1 338 487 L HD1 H 1 0.778 1 339 487 L HD2 H 1 0.829 1 340 487 L N N 15 121.648 1 341 489 S CA C 13 57.732 1 342 489 S CB C 13 63.976 1 343 489 S CO C 13 175.307 1 344 489 S H H 1 9.146 1 345 489 S N N 15 117.954 1 346 490 V CA C 13 59.96 1 347 490 V CB C 13 36.725 1 348 490 V CG1 C 13 21.034 1 349 490 V CG2 C 13 23.035 1 350 490 V CO C 13 175.445 1 351 490 V H H 1 7.378 1 352 490 V HG1 H 1 0.555 1 353 490 V HG2 H 1 0.885 1 354 490 V N N 15 118.616 1 355 491 H CA C 13 56.457 1 356 491 H CB C 13 33.175 1 357 491 H CO C 13 172.689 1 358 491 H H H 1 8.824 1 359 491 H N N 15 124.16 1 360 492 A CA C 13 48.933 1 361 492 A CB C 13 20.637 1 362 492 A CO C 13 175.149 1 363 492 A H H 1 7.475 1 364 492 A N N 15 129.587 1 365 493 F CA C 13 57.443 1 366 493 F CB C 13 40.032 1 367 493 F H H 1 8.075 1 368 493 F N N 15 120.292 1 369 494 N CA C 13 52.612 1 370 494 N CB C 13 39.014 1 371 494 N CO C 13 177.042 1 372 494 N H H 1 8.614 1 373 494 N N N 15 116.324 1 374 495 T CA C 13 65.108 1 375 495 T CB C 13 67.954 1 376 495 T H H 1 8.712 1 377 495 T N N 15 111.881 1 378 496 F CA C 13 57.589 1 379 496 F CB C 13 38.722 1 380 496 F CO C 13 176.791 1 381 496 F H H 1 8.441 1 382 496 F N N 15 120.392 1 383 497 F CA C 13 61.468 1 384 497 F CB C 13 38.442 1 385 497 F CO C 13 175.62 1 386 497 F H H 1 7.951 1 387 497 F N N 15 121.386 1 388 498 F CA C 13 62.45 1 389 498 F CB C 13 37.649 1 390 498 F CO C 13 176.151 1 391 498 F H H 1 10.059 1 392 498 F N N 15 120.473 1 393 499 T CA C 13 65.751 1 394 499 T CB C 13 68.656 1 395 499 T H H 1 7.099 1 396 499 T N N 15 111.797 1 397 500 K CA C 13 58.082 1 398 500 K CB C 13 30.293 1 399 500 K CO C 13 177.17 1 400 500 K H H 1 7.805 1 401 500 K N N 15 122.907 1 402 501 L CA C 13 56.922 1 403 501 L CB C 13 40.32 1 404 501 L CD1 C 13 21.344 1 405 501 L CD2 C 13 26.13 1 406 501 L H H 1 8.04 1 407 501 L HD1 H 1 0.619 1 408 501 L HD2 H 1 0.269 1 409 501 L N N 15 120.722 1 410 502 K CA C 13 58.359 1 411 502 K CB C 13 31.117 1 412 502 K CO C 13 177.542 1 413 502 K H H 1 8.113 1 414 502 K N N 15 117.113 1 415 503 T CA C 13 63.65 1 416 503 T CB C 13 69.641 1 417 503 T CO C 13 175.362 1 418 503 T H H 1 7.521 1 419 503 T N N 15 108.626 1 420 504 A CA C 13 51.681 1 421 504 A CB C 13 19.982 1 422 504 A CO C 13 177.923 1 423 504 A H H 1 8.417 1 424 504 A N N 15 124.229 1 425 505 G CA C 13 44.062 1 426 505 G CO C 13 173.703 1 427 505 G H H 1 7.404 1 428 505 G N N 15 108.216 1 429 506 Y CA C 13 61.372 1 430 506 Y CB C 13 38.185 1 431 506 Y CO C 13 177.707 1 432 506 Y H H 1 8.506 1 433 506 Y N N 15 118.015 1 434 507 Q CA C 13 58.073 1 435 507 Q CB C 13 26.321 1 436 507 Q CO C 13 177.318 1 437 507 Q H H 1 8.677 1 438 507 Q N N 15 113.949 1 439 508 A CA C 13 53.059 1 440 508 A CB C 13 19.41 1 441 508 A CO C 13 178.474 1 442 508 A H H 1 7.193 1 443 508 A N N 15 117.81 1 444 509 V CA C 13 59.584 1 445 509 V CB C 13 32.636 1 446 509 V CG1 C 13 19.036 1 447 509 V CG2 C 13 20.077 1 448 509 V CO C 13 178.833 1 449 509 V H H 1 6.99 1 450 509 V HG1 H 1 0.152 1 451 509 V HG2 H 1 0.505 1 452 509 V N N 15 104.928 1 453 510 K CA C 13 59.235 1 454 510 K CB C 13 30.396 1 455 510 K CO C 13 178.002 1 456 510 K H H 1 7.252 1 457 510 K N N 15 126.565 1 458 511 R CA C 13 56.969 1 459 511 R CB C 13 28.393 1 460 511 R H H 1 8.593 1 461 511 R N N 15 116.236 1 462 512 W CA C 13 59.154 1 463 512 W CB C 13 27.825 1 464 512 W CO C 13 178.179 1 465 512 W H H 1 8.477 1 466 512 W HE1 H 1 10.293 1 467 512 W N N 15 120.092 1 468 512 W NE1 N 15 129.338 1 469 513 T CA C 13 60 1 470 513 T CB C 13 65.562 1 471 513 T CO C 13 174.181 1 472 513 T H H 1 7.356 1 473 513 T N N 15 105.836 1 474 514 K CA C 13 59.285 1 475 514 K CB C 13 31.488 1 476 514 K CO C 13 177.271 1 477 514 K H H 1 7.187 1 478 514 K N N 15 120.77 1 479 515 K CA C 13 55.075 1 480 515 K CB C 13 31.34 1 481 515 K CO C 13 175.55 1 482 515 K H H 1 8.52 1 483 515 K N N 15 115.267 1 484 516 V CA C 13 60.315 1 485 516 V CB C 13 34.794 1 486 516 V CG1 C 13 22.213 1 487 516 V CG2 C 13 19.431 1 488 516 V CO C 13 173.373 1 489 516 V H H 1 7.346 1 490 516 V HG1 H 1 1.035 1 491 516 V HG2 H 1 0.828 1 492 516 V N N 15 118.521 1 493 517 D CA C 13 50.719 1 494 517 D CB C 13 39.298 1 495 517 D CO C 13 178.171 1 496 517 D H H 1 8.502 1 497 517 D N N 15 124.325 1 498 518 V CA C 13 64.12 1 499 518 V CB C 13 30.53 1 500 518 V CG1 C 13 21.974 1 501 518 V CG2 C 13 17.74 1 502 518 V CO C 13 173.205 1 503 518 V H H 1 8.909 1 504 518 V HG1 H 1 0.709 1 505 518 V HG2 H 1 0.246 1 506 518 V N N 15 121.419 1 507 519 F CA C 13 57.9 1 508 519 F CB C 13 36.872 1 509 519 F CO C 13 176.5 1 510 519 F H H 1 7.223 1 511 519 F N N 15 110.893 1 512 520 S CB C 13 64.082 1 513 520 S CO C 13 173.635 1 514 520 S H H 1 7.457 1 515 520 S N N 15 113.527 1 516 521 V CA C 13 58.421 1 517 521 V CB C 13 33.049 1 518 521 V CG1 C 13 21.474 1 519 521 V CG2 C 13 19.203 1 520 521 V CO C 13 174.363 1 521 521 V H H 1 6.675 1 522 521 V HG1 H 1 0.677 1 523 521 V HG2 H 1 0.736 1 524 521 V N N 15 114.244 1 525 522 D CA C 13 57.671 1 526 522 D CB C 13 42.241 1 527 522 D H H 1 8.177 1 528 522 D N N 15 120.102 1 529 523 I CA C 13 59.234 1 530 523 I H H 1 8.209 1 531 523 I N N 15 117.31 1 532 524 L CA C 13 51.912 1 533 524 L CB C 13 42.117 1 534 524 L CD1 C 13 24.261 2 535 524 L CD2 C 13 24.458 2 536 524 L H H 1 9.357 1 537 524 L HD1 H 1 0.826 2 538 524 L HD2 H 1 0.873 2 539 524 L N N 15 121.905 1 540 525 L CA C 13 53.109 1 541 525 L CB C 13 43.667 1 542 525 L CD1 C 13 27.473 1 543 525 L CD2 C 13 23.613 1 544 525 L H H 1 8.708 1 545 525 L HD1 H 1 0.737 1 546 525 L HD2 H 1 0.713 1 547 525 L N N 15 120.45 1 548 526 V CA C 13 59.564 1 549 526 V CB C 13 32.337 1 550 526 V CG1 C 13 20.681 1 551 526 V CG2 C 13 19.401 1 552 526 V H H 1 8.925 1 553 526 V HG1 H 1 −0.236 1 554 526 V HG2 H 1 0.488 1 555 526 V N N 15 120.847 1 556 528 I CA C 13 60.95 1 557 528 I CB C 13 39.801 1 558 528 I H H 1 8.737 1 559 528 I N N 15 125.023 1 560 529 H CA C 13 50.979 1 561 529 H CB C 13 29.375 1 562 529 H CO C 13 174.067 1 563 529 H H H 1 9.036 1 564 529 H N N 15 129.849 1 565 530 L CA C 13 52.799 1 566 530 L CB C 13 41.01 1 567 530 L CD1 C 13 25.841 1 568 530 L CD2 C 13 23.767 1 569 530 L CO C 13 176.318 1 570 530 L H H 1 8.525 1 571 530 L HD1 H 1 0.874 1 572 530 L HD2 H 1 0.77 1 573 530 L N N 15 130.501 1 574 531 G CA C 13 46.001 1 575 531 G CO C 13 174.79 1 576 531 G H H 1 8.157 1 577 531 G N N 15 115.336 1 578 532 V CA C 13 61.094 1 579 532 V CB C 13 30.784 1 580 532 V CG1 C 13 20.964 1 581 532 V CG2 C 13 18.117 1 582 532 V CO C 13 175.461 1 583 532 V H H 1 8.198 1 584 532 V HG1 H 1 0.532 1 585 532 V HG2 H 1 0.584 1 586 532 V N N 15 119.81 1 587 533 H CA C 13 55.28 1 588 533 H CB C 13 32.996 1 589 533 H CO C 13 174.498 1 590 533 H H H 1 7.803 1 591 533 H N N 15 121.771 1 592 534 W CA C 13 55.915 1 593 534 W CB C 13 32.49 1 594 534 W H H 1 6.407 1 595 534 W HE1 H 1 9.377 1 596 534 W N N 15 125.3 1 597 534 W NE1 N 15 128.192 1 598 535 C CA C 13 56.548 1 599 535 C CB C 13 30.615 1 600 535 C CO C 13 171.965 1 601 535 C H H 1 9.461 1 602 535 C N N 15 117.22 1 603 536 L CA C 13 54.008 1 604 536 L CB C 13 46.487 1 605 536 L CD1 C 13 22.301 1 606 536 L CD2 C 13 26.282 1 607 536 L H H 1 7.905 1 608 536 L HD1 H 1 0.679 1 609 536 L HD2 H 1 0.597 1 610 536 L N N 15 120.825 1 611 537 A CA C 13 49.576 1 612 537 A CB C 13 20.964 1 613 537 A H H 1 8.835 1 614 537 A N N 15 126.773 1 615 538 V CA C 13 60.449 1 616 538 V CB C 13 35.413 1 617 538 V CG1 C 13 21.698 1 618 538 V CG2 C 13 21.913 1 619 538 V CO C 13 174.727 1 620 538 V H H 1 9.071 1 621 538 V HG1 H 1 0.87 1 622 538 V HG2 H 1 0.809 1 623 538 V N N 15 119.546 1 624 539 V CA C 13 60.873 1 625 539 V CB C 13 32.053 1 626 539 V CG1 C 13 20.502 1 627 539 V CG2 C 13 19.475 1 628 539 V H H 1 9.402 1 629 539 V HG1 H 1 0.441 1 630 539 V HG2 H 1 0.881 1 631 539 V N N 15 130.501 1 632 540 D CA C 13 51.922 1 633 540 D CB C 13 41.816 1 634 540 D H H 1 8.954 1 635 540 D N N 15 126.546 1 636 541 F CA C 13 62.022 1 637 541 F CB C 13 39.187 1 638 541 F H H 1 9.479 1 639 541 F N N 15 123.936 1 640 542 R CA C 13 57.3 1 641 542 R CB C 13 28.607 1 642 542 R CO C 13 179.074 1 643 542 R H H 1 8.714 1 644 542 R N N 15 117.561 1 645 543 K CA C 13 54.808 1 646 543 K CB C 13 33.288 1 647 543 K CO C 13 175.221 1 648 543 K H H 1 6.749 1 649 543 K N N 15 114.224 1 650 544 K CA C 13 55.562 1 651 544 K CB C 13 27.641 1 652 544 K CO C 13 175.247 1 653 544 K H H 1 7.423 1 654 544 K N N 15 115.776 1 655 545 N CA C 13 51.023 1 656 545 N CB C 13 42.123 1 657 545 N CO C 13 173.859 1 658 545 N H H 1 7.23 1 659 545 N N N 15 113.498 1 660 546 I CA C 13 61.517 1 661 546 I H H 1 8.432 1 662 546 I N N 15 120.288 1 663 547 T CA C 13 60.921 1 664 547 T CB C 13 70.493 1 665 547 T H H 1 8.781 1 666 547 T N N 15 121.352 1 667 548 Y CA C 13 57.227 1 668 548 Y CB C 13 40.796 1 669 548 Y H H 1 8.727 1 670 548 Y N N 15 128.981 1 671 549 Y CB C 13 40.072 1 672 549 Y H H 1 9.079 1 673 549 Y N N 15 125.239 1 674 550 D CA C 13 52.549 1 675 550 D CB C 13 43.937 1 676 550 D CO C 13 177.444 1 677 550 D H H 1 8.116 1 678 550 D N N 15 123.169 1 679 551 S CA C 13 60.463 1 680 551 S CB C 13 62.962 1 681 551 S CO C 13 174.422 1 682 551 S H H 1 9.519 1 683 551 S N N 15 122.969 1 684 552 M CA C 13 54.901 1 685 552 M CB C 13 34.489 1 686 552 M CO C 13 178.972 1 687 552 M H H 1 9.32 1 688 552 M N N 15 122.574 1 689 553 G CA C 13 46.475 1 690 553 G CO C 13 175.385 1 691 553 G H H 1 7.89 1 692 553 G N N 15 109.507 1 693 554 G CA C 13 44.928 1 694 554 G CO C 13 171.658 1 695 554 G H H 1 7.51 1 696 554 G N N 15 107.555 1 697 555 I CA C 13 59.215 1 698 555 I CB C 13 37.955 1 699 555 I CO C 13 176.285 1 700 555 I H H 1 8.05 1 701 555 I N N 15 118.138 1 702 556 N CA C 13 50.688 1 703 556 N CB C 13 36.209 1 704 556 N H H 1 7.762 1 705 556 N N N 15 124.106 1 706 557 N CA C 13 55.66 1 707 557 N CB C 13 37.225 1 708 557 N H H 1 8.339 1 709 557 N N N 15 121.312 1 710 558 E CA C 13 59.21 1 711 558 E CB C 13 28.223 1 712 558 E H H 1 8.531 1 713 558 E N N 15 120.721 1 714 559 A CA C 13 55.072 1 715 559 A CB C 13 17.209 1 716 559 A CO C 13 179.228 1 717 559 A H H 1 7.491 1 718 559 A N N 15 120.499 1 719 560 C CA C 13 61.861 1 720 560 C CB C 13 26.362 1 721 560 C CO C 13 176.152 1 722 560 C H H 1 6.803 1 723 560 C N N 15 111.946 1 724 561 R CA C 13 59.736 1 725 561 R CB C 13 29.122 1 726 561 R CO C 13 179.458 1 727 561 R H H 1 8.077 1 728 561 R N N 15 120.238 1 729 562 I CA C 13 64.773 1 730 562 I CB C 13 36.965 1 731 562 I CO C 13 179.283 1 732 562 I H H 1 8.583 1 733 562 I N N 15 120.588 1 734 563 L CA C 13 56.989 1 735 563 L CB C 13 41.139 1 736 563 L CD1 C 13 26.07 1 737 563 L CD2 C 13 22.794 1 738 563 L CO C 13 177.668 1 739 563 L H H 1 7.58 1 740 563 L HD1 H 1 0.714 1 741 563 L HD2 H 1 0.791 1 742 563 L N N 15 120.645 1 743 564 L CA C 13 57.863 1 744 564 L CB C 13 40.586 1 745 564 L CD1 C 13 23.029 1 746 564 L CD2 C 13 25.722 1 747 564 L H H 1 7.989 1 748 564 L HD1 H 1 0.503 1 749 564 L HD2 H 1 0.873 1 750 564 L N N 15 122.277 1 751 565 Q CB C 13 27.023 1 752 565 Q CO C 13 178.554 1 753 565 Q H H 1 7.945 1 754 565 Q N N 15 116.169 1 755 566 Y CA C 13 61.262 1 756 566 Y CB C 13 36.89 1 757 566 Y H H 1 8.147 1 758 566 Y N N 15 121.414 1 759 567 L CA C 13 57.69 1 760 567 L CB C 13 39.693 1 761 567 L CD1 C 13 26.027 1 762 567 L CD2 C 13 21.698 1 763 567 L H H 1 7.756 1 764 567 L HD1 H 1 0.298 1 765 567 L HD2 H 1 0.574 1 766 567 L N N 15 118.927 1 767 568 K CA C 13 59.666 1 768 568 K CB C 13 31.072 1 769 568 K CO C 13 180.159 1 770 568 K H H 1 7.436 1 771 568 K N N 15 116.321 1 112 569 Q CA C 13 58.376 1 773 569 Q CB C 13 26.837 1 774 569 Q CO C 13 178.121 1 775 569 Q H H 1 7.71 1 776 569 Q N N 15 119.281 1 111 570 E CA C 13 57.248 1 778 570 E CB C 13 27.85 1 779 570 E CO C 13 178.511 1 780 570 E H H 1 8.874 1 781 570 E N N 15 123.757 1 782 571 S CA C 13 61.824 1 783 571 S CB C 13 63.591 1 784 571 S H H 1 8.112 1 785 571 S N N 15 113.072 1 786 572 I CA C 13 64.046 1 787 572 I CB C 13 36.762 1 788 572 I CO C 13 178.898 1 789 572 I H H 1 7.085 1 790 572 I N N 15 120.025 1 791 573 D CA C 13 58.589 1 792 573 D CB C 13 45.295 1 793 573 D H H 1 8.267 1 794 573 D N N 15 119.596 1 795 574 K CA C 13 55.261 1 796 574 K H H 1 8.537 1 797 574 K N N 15 110.111 1 798 575 K CA C 13 53.6 1 799 575 K H H 1 7.814 1 800 575 K N N 15 114.921 1 801 580 D CA C 13 53.108 1 802 580 D CO C 13 175.974 1 803 580 D H H 1 8.008 1 804 580 D N N 15 128.24 1 805 581 T CA C 13 61.607 1 806 581 T CB C 13 67.992 1 807 581 T CO C 13 176.37 1 808 581 T H H 1 8.005 1 809 581 T N N 15 114.488 1 810 582 N CA C 13 55.671 1 811 582 N CB C 13 37.704 1 812 582 N CO C 13 177.026 1 813 582 N H H 1 8.559 1 814 582 N N N 15 124.721 1 815 583 G CA C 13 45.005 1 816 583 G CO C 13 174.64 1 817 583 G H H 1 8.964 1 818 583 G N N 15 113.066 1 819 584 W CA C 13 58.735 1 820 584 W CB C 13 27.343 1 821 584 W CO C 13 177.331 1 822 584 W H H 1 7.891 1 823 584 W HE1 H 1 10.207 1 824 584 W N N 15 120.551 1 825 584 W NE1 N 15 130.072 1 826 585 Q CA C 13 54.336 1 827 585 Q CB C 13 32.617 1 828 585 Q CO C 13 173.396 1 829 585 Q H H 1 8.32 1 830 585 Q N N 15 120.47 1 831 586 L CA C 13 52.787 1 832 586 L CB C 13 41.572 1 833 586 L CD1 C 13 24.58 1 834 586 L CD2 C 13 24.177 1 835 586 L CO C 13 176.073 1 836 586 L H H 1 8.257 1 837 586 L HD1 H 1 0.875 1 838 586 L HD2 H 1 1.06 1 839 586 L N N 15 122.471 1 840 587 F CA C 13 56.274 1 841 587 F CB C 13 41.98 1 842 587 F CO C 13 174.71 1 843 587 F H H 1 9.007 1 844 587 F N N 15 119.628 1 845 588 S CA C 13 57.584 1 846 588 S CB C 13 64.741 1 847 588 S CO C 13 174.522 1 848 588 S H H 1 8.55 1 849 588 S N N 15 115.508 1 850 589 K CA C 13 54.504 1 851 589 K CB C 13 30.677 1 852 589 K CO C 13 176.928 1 853 589 K H H 1 8.375 1 854 589 K N N 15 123.879 1 855 590 K CA C 13 55.59 1 856 590 K CB C 13 32.654 1 857 590 K CO C 13 178.646 1 858 590 K H H 1 9.16 1 859 590 K N N 15 124.442 1 860 591 S CA C 13 60.73 1 861 591 S CB C 13 62.469 1 862 591 S CO C 13 175.07 1 863 591 S H H 1 8.771 1 864 591 S N N 15 116.878 1 865 592 Q CA C 13 56.481 1 866 592 Q CB C 13 27.319 1 867 592 Q CO C 13 177.119 1 868 592 Q H H 1 7.787 1 869 592 Q N N 15 114.421 1 870 593 E CA C 13 56.788 1 871 593 E CB C 13 31.514 1 872 593 E CO C 13 176.185 1 873 593 E H H 1 8.147 1 874 593 E N N 15 116.571 1 875 594 I CA C 13 57.233 1 876 594 I CB C 13 39.464 1 877 594 I H H 1 7.099 1 878 594 I N N 15 111.638 1 879 596 Q CA C 13 52.641 1 880 596 Q CB C 13 31.367 1 881 596 Q CO C 13 176.998 1 882 596 Q H H 1 8.568 1 883 596 Q N N 15 119.776 1 884 597 Q CA C 13 53.866 1 885 597 Q CB C 13 28.195 1 886 597 Q CO C 13 175.677 1 887 597 Q H H 1 8.67 1 888 597 Q N N 15 118.265 1 889 598 M CA C 13 55.496 1 890 598 M CB C 13 33.978 1 891 598 M CO C 13 175.887 1 892 598 M H H 1 9.45 1 893 598 M N N 15 118.496 1 894 599 N CA C 13 51.768 1 895 599 N CB C 13 39.114 1 896 599 N H H 1 7.565 1 897 599 N N N 15 117.184 1 898 600 G H H 1 9.054 1 899 600 G N N 15 114.081 1 900 601 S CA C 13 58.424 1 901 601 S CB C 13 60.647 1 902 601 S H H 1 7.855 1 903 601 S N N 15 114.866 1 904 602 D CA C 13 55.181 1 905 602 D CB C 13 40.81 1 906 602 D CO C 13 178.539 1 907 602 D H H 1 7.257 1 908 602 D N N 15 118.282 1 909 603 C CA C 13 60.678 1 910 603 C CB C 13 28.27 1 911 603 C CO C 13 175.783 1 912 603 C H H 1 7.715 1 913 603 C N N 15 121.968 1 914 604 G CA C 13 46.862 1 915 604 G CO C 13 175.076 1 916 604 G H H 1 8.736 1 917 604 G N N 15 109.643 1 918 605 M CA C 13 54.421 1 919 605 M CB C 13 28.885 1 920 605 M CO C 13 178.831 1 921 605 M H H 1 6.973 1 922 605 M N N 15 118.381 1 923 606 F CA C 13 63.186 1 924 606 F CB C 13 37.346 1 925 606 F CO C 13 175.928 1 926 606 F H H 1 8.265 1 927 606 F N N 15 118.962 1 928 607 A CA C 13 55.826 1 929 607 A CB C 13 15.841 1 930 607 A CO C 13 179.705 1 931 607 A H H 1 7.745 1 932 607 A N N 15 118.262 1 933 608 C CA C 13 64.555 1 934 608 C CB C 13 26.489 1 935 608 C CO C 13 176.344 1 936 608 C H H 1 7.15 1 937 608 C N N 15 111.327 1 938 609 K CA C 13 55.983 1 939 609 K CB C 13 28.003 1 940 609 K CO C 13 180.898 1 941 609 K H H 1 8.114 1 942 609 K N N 15 117.546 1 943 610 Y CA C 13 58.081 1 944 610 Y CB C 13 36.268 1 945 610 Y CO C 13 177.942 1 946 610 Y H H 1 9.584 1 947 610 Y N N 15 121.342 1 948 611 A CA C 13 55.284 1 949 611 A CB C 13 17.36 1 950 611 A CO C 13 179.296 1 951 611 A H H 1 7.3 1 952 611 A N N 15 118.235 1 953 612 D CA C 13 57.496 1 954 612 D CB C 13 40.809 1 955 612 D CO C 13 177.083 1 956 612 D H H 1 8.285 1 957 612 D N N 15 119.051 1 958 613 C CA C 13 64.249 1 959 613 C CB C 13 26.401 1 960 613 C CO C 13 177.033 1 961 613 C H H 1 7.283 1 962 613 C N N 15 114.15 1 963 614 I CA C 13 64.188 1 964 614 I CB C 13 38.287 1 965 614 I CO C 13 180.531 1 966 614 I H H 1 8.523 1 967 614 I N N 15 119.103 1 968 615 T CB C 13 67.658 1 969 615 T CO C 13 174.525 1 970 615 T H H 1 8.251 1 971 615 T N N 15 108.325 1 972 616 K CA C 13 55.419 1 973 616 K CB C 13 31.971 1 974 616 K CO C 13 175.509 1 975 616 K H H 1 7.204 1 976 616 K N N 15 118.784 1 977 617 D CA C 13 55.028 1 978 617 D CB C 13 38.871 1 979 617 D CO C 13 174.882 1 980 617 D H H 1 7.982 1 981 617 D N N 15 117.696 1 982 618 R CA C 13 51.906 1 983 618 R CB C 13 30.614 1 984 618 R CO C 13 173.638 1 985 618 R H H 1 7.893 1 986 618 R N N 15 116.707 1 987 620 I CA C 13 62.112 1 988 620 I CB C 13 35.731 1 989 620 I CO C 13 177.018 1 990 620 I H H 1 8.465 1 991 620 I N N 15 121.915 1 992 621 N CA C 13 52.404 1 993 621 N CB C 13 38.108 1 994 621 N H H 1 7.952 1 995 621 N N N 15 126.058 1 996 622 F CA C 13 54.399 1 997 622 F CB C 13 41.066 1 998 622 F CO C 13 173.442 1 999 622 F H H 1 6.573 1 1000 622 F N N 15 114.01 1 1001 623 T CA C 13 59.922 1 1002 623 T CB C 13 73.255 1 1003 623 T H H 1 11.019 1 1004 623 T N N 15 112.666 1 1005 624 Q CA C 13 57.991 1 1006 624 Q CB C 13 28.115 1 1007 624 Q CO C 13 177.817 1 1008 624 Q H H 1 9.841 1 1009 624 Q N N 15 118.693 1 1010 625 Q CA C 13 57.772 1 1011 625 Q CB C 13 27.232 1 1012 625 Q CO C 13 177.118 1 1013 625 Q H H 1 8.35 1 1014 625 Q N N 15 118.528 1 1015 626 H CA C 13 59.023 1 1016 626 H CB C 13 32.018 1 1017 626 H CO C 13 175.195 1 1018 626 H H H 1 7.69 1 1019 626 H N N 15 116.12 1 1020 627 M CA C 13 58.703 1 1021 627 M CB C 13 29.282 1 1022 627 M CO C 13 175.481 1 1023 627 M H H 1 7.581 1 1024 627 M N N 15 117.619 1 1025 629 Y CA C 13 59.898 1 1026 629 Y CB C 13 36.935 1 1027 629 Y CO C 13 176.303 1 1028 629 Y H H 1 7.455 1 1029 629 Y N N 15 119.231 1 1030 630 F CA C 13 57.548 1 1031 630 F CO C 13 179.707 1 1032 630 F H H 1 8.72 1 1033 630 F N N 15 118.661 1 1034 631 R CA C 13 59.879 1 1035 631 R CB C 13 29.774 1 1036 631 R CO C 13 177.119 1 1037 631 R H H 1 8.743 1 1038 631 R N N 15 121.276 1 1039 632 K CB C 13 32.019 1 1040 632 K CO C 13 178.005 1 1041 632 K H H 1 7.027 1 1042 632 K N N 15 115.472 1 1043 633 R CA C 13 59.11 1 1044 633 R CB C 13 30.377 1 1045 633 R H H 1 8.483 1 1046 633 R N N 15 116.56 1 1047 634 M CA C 13 57.95 1 1048 634 M CB C 13 31.937 1 1049 634 M H H 1 8.212 1 1050 634 M N N 15 116.933 1 1051 635 V CA C 13 66.729 1 1052 635 V CB C 13 30.848 1 1053 635 V CG1 C 13 22.182 1 1054 635 V CG2 C 13 24.307 1 1055 635 V CO C 13 176.781 1 1056 635 V H H 1 7.401 1 1057 635 V HG1 H 1 0.511 1 1058 635 V HG2 H 1 1.078 1 1059 635 V N N 15 117.807 1 1060 636 W CA C 13 63.021 1 1061 636 W CB C 13 28.855 1 1062 636 W CO C 13 177.991 1 1063 636 W H H 1 6.945 1 1064 636 W HE1 H 1 10.208 1 1065 636 W N N 15 117.761 1 1066 636 W NE1 N 15 131.254 1 1067 637 E CA C 13 59.634 1 1068 637 E CB C 13 29.54 1 1069 637 E CO C 13 179.549 1 1070 637 E H H 1 8.942 1 1071 637 E N N 15 118.312 1 1072 638 I CA C 13 65.061 1 1073 638 I CB C 13 36.564 1 1074 638 I CO C 13 178.793 1 1075 638 I H H 1 8.516 1 1076 638 I N N 15 118.151 1 1077 639 L CA C 13 57.489 1 1078 639 L CB C 13 40.751 1 1079 639 L CD1 C 13 25.065 1 1080 639 L CD2 C 13 22.821 1 1081 639 L H H 1 8.021 1 1082 639 L HD1 H 1 0.577 1 1083 639 L HD2 H 1 0.498 1 1084 639 L N N 15 119.857 1 1085 640 H CA C 13 55.633 1 1086 640 H CB C 13 26.845 1 1087 640 H H H 1 7.758 1 1088 640 H N N 15 112.218 1 1089 641 R CA C 13 56.955 1 1090 641 R CB C 13 25.944 1 1091 641 R CO C 13 174.773 1 1092 641 R H H 1 7.879 1 1093 641 R N N 15 122.564 1 1094 642 K CA C 13 54.648 1 1095 642 K CB C 13 34.915 1 1096 642 K CO C 13 172.886 1 1097 642 K H H 1 8.22 1 1098 642 K N N 15 121.148 1 1099 643 L CA C 13 53.678 1 1100 643 L CB C 13 41.237 1 1101 643 L CD1 C 13 27.306 1 1102 643 L CD2 C 13 24.45 1 1103 643 L CO C 13 177.594 1 1104 643 L H H 1 8.136 1 1105 643 L HD1 H 1 0.395 1 1106 643 L HD2 H 1 0.572 1 1107 643 L N N 15 122.205 1 1108 644 L CA C 13 55.222 1 1109 644 L CB C 13 41.615 1 1110 644 L CO C 13 182.082 1 1111 644 L H H 1 8.863 1 1112 644 L N N 15 130.114 1 *referenced using DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) as the H-1 standard with IUPAC-IUB recommended chemical shift referencing ratios. See, Wishart, et al., “1H, 13C and 15N Chemical Shift Referencing in Biomolecular NMR,” J. Biomol. NMR 6: 135-140 (1995); and Markley et al., “Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids,”. Pure & Appl. Chem. 70: 117-142 (1998).

TABLE 4 SENP1 C603S-SUMO₁₋₉₂ NMR Chemical Shift Values. Chemical Shift Ambiguity Index Value Definitions The values other than 1 are used for those atoms with different chemical shifts that cannot be assigned to stereospecific atoms or to specific residues or chains. Index Value Definition 1 Unique (including isolated methyl protons germinal atoms, and geminal methyl groups with identical chemical shifts (e.g. ILE HD11, HD12, HD13 protons) 2 Ambiguity of geminal atoms or geminal methyl proton groups (e.g. ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, or LEU HD11, HD12, HD13 and HD21, HD22, HD23 methyl protons) 3 Aromatic atoms on opposite sides of symmetrical rings (e.g. TYR HE1 and HE2 protons) 4 Intraresidue ambiguities (e.g. LYS HG and HD protons or TRP HZ2 and HZ3 protons) 5 Interresidue ambiguities (LYS 12 vs. LYS 27) 6 Intermolecular ambiguities (e.g. ASP 31 CA in monomer 1 and ASP 31 CA in monomer 2 of an asymmetrical homodimer, duplex DNA assignments, or other assignments that may apply to atoms in one or more molecule in the molecular assembly) 9 Ambiguous, specific ambiguity not defined Chemical Atom Residue Amino Atom Atom Iso- shift Unique- number number acid context type type (ppm)* ness 1 419 E H H 1 7.974 1 2 419 E N N 15 121.157 1 3 420 F H H 1 7.947 1 4 420 F N N 15 119.83 1 5 422 E H H 1 8.562 1 6 422 E N N 15 125.045 1 7 423 I H H 1 8.443 1 8 423 I N N 15 123.042 1 9 424 T H H 1 7.558 1 10 424 T N N 15 122.22 1 11 425 E H H 1 8.835 1 12 425 E N N 15 122.015 1 13 426 E H H 1 8.343 1 14 426 E N N 15 119.067 1 15 427 M H H 1 7.295 1 16 427 M N N 15 120.177 1 17 428 E H H 1 8.525 1 18 428 E N N 15 119.639 1 19 429 K H H 1 7.793 1 20 429 K N N 15 119.161 1 21 430 E H H 1 7.247 1 22 430 E N N 15 120.017 1 23 432 K H H 1 8.269 1 24 432 K N N 15 117.252 1 25 433 D H H 1 7.542 1 26 433 D N N 15 116.02 1 27 434 V CG1 C 13 22.744 1 28 434 V H H 1 7.456 1 29 434 V N N 15 115.391 1 32 434 V HG1 H 1 0.768 1 33 435 F H H 1 7.229 1 34 435 F N N 15 118.53 1 35 436 R H H 1 7.082 1 36 436 R N N 15 119.966 1 37 437 D H H 1 8.266 1 38 437 D N N 15 120.742 1 39 438 G H H 1 7.994 1 40 438 G N N 15 110.561 1 41 439 D H H 1 8.68 1 42 439 D N N 15 121.505 1 43 440 Q H H 1 8.954 1 44 440 Q N N 15 126.9 1 45 441 D H H 1 7.858 1 46 441 D N N 15 115.853 1 47 442 E H H 1 7.08 1 48 442 E N N 15 122.689 1 49 443 V CG1 C 13 21.595 1 50 443 V CG2 C 13 22.122 1 51 443 V H H 1 8.563 1 52 443 V N N 15 128.078 1 55 443 V HG1 H 1 0.731 1 58 443 V HG2 H 1 0.896 1 59 444 L CD1 C 13 27.192 1 60 444 L H H 1 8.928 1 61 444 L N N 15 128.342 1 64 444 L HD1 H 1 0.609 1 65 445 S H H 1 7.327 1 66 445 S N N 15 112.548 1 67 446 E H H 1 7.889 1 68 446 E N N 15 125.959 1 69 447 A H H 1 8.267 1 70 447 A N N 15 125.511 1 71 448 F H H 1 8.549 1 72 448 F N N 15 117.045 1 73 449 R H H 1 8.43 1 74 449 R N N 15 112.963 1 75 450 L CD1 C 13 26.152 1 76 450 L CD2 C 13 23.171 1 77 450 L H H 1 8.288 1 78 450 L N N 15 121.64 1 81 450 L HD1 H 1 0.918 1 84 450 L HD2 H 1 1.032 1 85 451 T H H 1 8.297 1 86 451 T N N 15 113.505 1 87 452 I H H 1 8.385 1 88 452 I N N 15 124.62 1 89 453 T H H 1 9.666 1 90 453 T N N 15 120.795 1 91 454 R H H 1 8.168 1 92 454 R N N 15 123.055 1 93 455 K H H 1 8.41 1 94 455 K N N 15 120.473 1 95 456 D H H 1 7.227 1 96 456 D N N 15 118.114 1 97 457 I H H 1 8.102 1 98 457 I N N 15 122.652 1 99 458 Q H H 1 7.821 1 100 458 Q N N 15 119.174 1 101 459 T H H 1 7.826 1 102 459 T N N 15 114.259 1 103 460 L CD1 C 13 26.138 1 104 460 L CD2 C 13 26.003 1 105 460 L H H 1 7.18 1 106 460 L N N 15 116.293 1 109 460 L HD1 H 1 0.845 1 112 460 L HD2 H 1 0.954 1 113 461 D H H 1 7.356 1 114 461 D N N 15 121.211 1 115 462 H H H 1 7.659 1 116 462 H N N 15 120.742 1 117 465 W H H 1 8.258 1 118 465 W N N 15 121.504 1 119 465 W HE1 H 1 9.997 1 120 465 W NE1 H 1 130.62 1 121 466 L CD1 C 13 25.625 1 122 466 L CD2 C 13 23.625 1 123 466 L H H 1 7.529 1 124 466 L N N 15 126.826 1 127 466 L HD1 H 1 0.684 1 130 466 L HD2 H 1 0.704 1 131 467 D H H 1 6.937 1 132 467 D N N 15 117.87 1 133 468 D H H 1 8.19 1 134 468 D N N 15 115.279 1 135 470 I H H 1 7.581 1 136 470 I N N 15 118.739 1 137 471 I H H 1 6.823 1 138 471 I N N 15 118.333 1 139 472 D H H 1 8.781 1 140 472 D N N 15 116.126 1 141 473 F H H 1 8.248 1 142 473 F N N 15 124.484 1 143 475 M H H 1 8.642 1 144 475 M N N 15 116.267 1 145 476 D H H 1 7.328 1 146 476 D N N 15 118.167 1 147 477 M H H 1 7.588 1 148 477 M N N 15 122.218 1 149 478 L CD1 C 13 27.574 1 150 478 L CD2 C 13 22.365 1 151 478 L H H 1 7.689 1 152 478 L N N 15 121.041 1 155 478 L HD1 H 1 0.707 1 158 478 L HD2 H 1 0.458 1 159 479 M H H 1 7.581 1 160 479 M N N 15 120.182 1 161 480 E H H 1 8.04 1 162 480 E N N 15 123.808 1 163 481 R H H 1 7.872 1 164 481 R N N 15 121.587 1 165 482 S H H 1 7.133 1 166 482 S N N 15 114.199 1 167 483 K H H 1 6.896 1 168 483 K N N 15 119.755 1 169 484 E H H 1 8.035 1 170 484 E N N 15 122.017 1 171 485 K H H 1 8.183 1 172 485 K N N 15 122.621 1 173 486 G H H 1 8.674 1 174 486 G N N 15 112.424 1 175 487 L CD1 C 13 25.976 1 176 487 L CD2 C 13 23.426 1 177 487 L H H 1 7.277 1 178 487 L N N 15 122.648 1 181 487 L HD1 H 1 0.802 1 184 487 L HD2 H 1 0.853 1 185 489 S H H 1 9.049 1 186 489 S N N 15 119.012 1 187 490 V CG1 C 13 21.236 1 188 490 V CG2 C 13 23.244 1 189 490 V H H 1 7.325 1 190 490 V N N 15 119.713 1 191 490 V HG1 H 1 0.583 1 196 490 V HG2 H 1 0.912 1 197 491 H H H 1 8.716 1 198 491 H N N 15 125.135 1 199 492 A H H 1 7.396 1 200 492 A N N 15 130.645 1 201 494 D H H 1 8.676 1 202 494 D N N 15 117.371 1 203 495 T H H 1 8.641 1 204 495 T N N 15 112.625 1 205 497 F H H 1 7.868 1 206 497 F N N 15 122.062 1 207 498 F H H 1 9.932 1 208 498 F N N 15 121.373 1 209 499 T H H 1 6.901 1 210 499 T N N 15 113.079 1 211 500 K H H 1 7.689 1 212 500 K N N 15 123.985 1 213 501 L CD1 C 13 21.431 1 214 501 L CD2 C 13 26.226 1 217 501 L HD1 H 1 0.589 1 220 501 L HD2 H 1 0.244 1 221 502 K H H 1 8.034 1 222 502 K N N 15 118.063 1 223 503 T H H 1 7.409 1 224 503 T N N 15 109.905 1 225 504 A H H 1 8.332 1 226 504 A N N 15 125.057 1 227 505 G H H 1 7.268 1 228 505 G N N 15 109.015 1 229 506 Y H H 1 8.399 1 230 506 Y N N 15 118.799 1 231 507 Q H H 1 8.555 1 232 507 Q N N 15 114.738 1 233 508 A H H 1 7.049 1 234 508 A N N 15 118.763 1 235 509 V CG1 C 13 19.211 1 236 509 V CG2 C 13 20.045 1 237 509 V H H 1 6.759 1 238 509 V N N 15 105.237 1 241 509 V HG1 H 1 0.198 1 244 509 V HG2 H 1 0.47 1 245 510 K H H 1 7.119 1 246 510 K N N 15 128.018 1 247 511 R H H 1 8.684 1 248 511 R N N 15 117.231 1 249 512 W H H 1 8.542 1 250 512 W N N 15 120.625 1 251 512 W HE1 H 1 9.942 1 252 512 W NE1 H 1 130.341 1 253 513 T H H 1 7.069 1 254 513 T N N 15 106.003 1 255 514 K H H 1 7.238 1 256 514 K N N 15 121.358 1 257 515 K H H 1 8.487 1 258 515 K N N 15 116.476 1 259 516 V CG1 C 13 22.32 1 260 516 V CG2 C 13 19.555 1 261 516 V H H 1 7.299 1 262 516 V N N 15 119.473 1 265 516 V HG1 H 1 1.018 1 268 516 V HG2 H 1 0.836 1 269 517 D H H 1 8.389 1 270 517 D N N 15 124.991 1 271 518 V CG1 C 13 21.972 1 272 518 V CG2 C 13 17.937 1 273 518 V H H 1 8.864 1 274 518 V N N 15 122.483 1 277 518 V HG1 H 1 0.73 1 280 518 V HG2 H 1 0.268 1 281 519 F H H 1 7.156 1 282 519 F N N 15 111.705 1 283 520 S H H 1 7.418 1 284 520 S N N 15 114.604 1 285 521 V CG1 C 13 21.606 1 286 521 V CG2 C 13 19.292 1 287 521 V H H 1 6.57 1 288 521 V N N 15 114.783 1 291 521 V HG1 H 1 0.698 1 294 521 V HG2 H 1 0.757 1 295 522 D H H 1 8.102 1 296 522 D N N 15 121.096 1 297 523 I H H 1 8.117 1 298 523 I N N 15 118.339 1 299 524 L CD1 C 13 24.558 2 300 524 L CD2 C 13 24.558 2 301 524 L H H 1 9.253 1 302 524 L N N 15 122.892 1 305 524 L HG1 H 1 0.841 2 308 524 L HG2 H 1 0.841 2 309 525 L CD1 C 13 27.708 2 310 525 L CD2 C 13 23.849 1 311 525 L H H 1 8.636 1 312 525 L N N 15 121.574 1 315 525 L HD1 H 1 0.775 1 318 525 L HD2 H 1 0.735 1 319 526 V CG1 C 13 20.514 1 320 526 V CG2 C 13 19.367 1 323 526 V HG1 H 1 −0.557 1 326 526 V HG2 H 1 0.332 1 327 528 I H H 1 8.599 1 328 528 I N N 15 125.838 1 329 529 H H H 1 9.049 1 330 529 H N N 15 130.138 1 331 530 L CD1 C 13 25.956 1 332 530 L CD2 C 13 23.945 1 333 530 L H H 1 8.536 1 334 530 L N N 15 131.927 1 337 530 L HD1 H 1 0.891 1 340 530 L HD2 H 1 0.759 1 341 531 G H H 1 8 1 342 531 G N N 15 116.007 1 343 532 V CG1 C 13 20.988 1 344 532 V CG2 C 13 17.895 1 347 532 V HG1 H 1 0.507 1 350 532 V HG2 H 1 0.396 1 351 533 H H H 1 7.652 1 352 533 H N N 15 123.973 1 353 534 W H H 1 7.787 1 354 534 W N N 15 125.281 1 355 534 W HE1 H 1 9.36 1 356 534 W NE1 H 1 128.566 1 357 535 C H H 1 9.374 1 358 535 C N N 15 118.493 1 359 536 L CD1 C 13 22.488 1 360 536 L CD2 C 13 26.383 1 363 536 L HD1 H 1 0.708 1 366 536 L HD2 H 1 0.622 1 367 537 A H H 1 8.735 1 368 537 A N N 15 127.969 1 369 538 V CG1 C 13 21.782 1 370 538 V CG2 C 13 22.071 1 371 538 V H H 1 8.971 1 372 538 V N N 15 120.496 1 375 538 V HG1 H 1 0.885 1 378 538 V HG2 H 1 0.831 1 379 539 V CG1 C 13 20.583 1 380 539 V CG2 C 13 19.613 1 381 539 V H H 1 9.299 1 382 539 V N N 15 131.537 1 385 539 V HG1 H 1 0.452 1 388 539 V HG2 H 1 0.894 1 389 540 D H H 1 8.849 1 390 540 D N N 15 127.571 1 391 541 F H H 1 9.394 1 392 541 F N N 15 125.014 1 393 542 R H H 1 8.636 1 394 542 R N N 15 118.378 1 395 543 K H H 1 6.667 1 396 543 K N N 15 115.058 1 397 544 K H H 1 7.337 1 398 544 K N N 15 116.66 1 399 545 D H H 1 7.143 1 400 545 D N N 15 114.477 1 401 546 I H H 1 8.348 1 402 546 I N N 15 121.213 1 403 547 T H H 1 8.68 1 404 547 T N N 15 122.503 1 405 548 Y H H 1 8.599 1 406 548 Y N N 15 129.81 1 407 549 Y H H 1 8.988 1 408 549 Y N N 15 126.841 1 409 550 D H H 1 7.986 1 410 550 D N N 15 124.111 1 411 551 S H H 1 9.257 1 412 551 S N N 15 123.506 1 413 552 M H H 1 9.118 1 414 552 M N N 15 122.883 1 415 553 G H H 1 7.765 1 416 553 G N N 15 110.571 1 417 554 G H H 1 7.593 1 418 554 G N N 15 108.922 1 419 555 I H H 1 7.986 1 420 555 I N N 15 119.104 1 421 556 D H H 1 7.676 1 422 556 D N N 15 125.208 1 423 557 D H H 1 8.221 1 424 557 D N N 15 122.087 1 425 558 E H H 1 8.444 1 426 558 E N N 15 121.771 1 427 559 A H H 1 7.431 1 428 559 A N N 15 121.623 1 429 560 C H H 1 6.749 1 430 560 C N N 15 112.908 1 431 561 R H H 1 7.935 1 432 561 R N N 15 121.153 1 433 562 I H H 1 8.494 1 434 562 I N N 15 121.654 1 435 563 L CD1 C 13 26.251 1 436 563 L H H 1 7.51 1 437 563 L N N 15 121.675 1 440 563 L HD1 H 1 0.705 1 441 564 L CD1 C 13 23.21 1 442 564 L CD2 C 13 25.851 1 443 564 L H H 1 7.927 1 444 564 L N N 15 123.395 1 447 564 L HD1 H 1 0.527 1 450 564 L HD2 H 1 0.857 1 451 565 Q H H 1 7.814 1 452 565 Q N N 15 117.094 1 453 566 Y H H 1 8.072 1 454 566 Y N N 15 122.424 1 455 567 L CD1 C 13 26.248 1 456 567 L H H 1 7.659 1 457 567 L N N 15 119.901 1 460 567 L HD1 H 1 0.295 1 461 568 K H H 1 7.303 1 462 568 K N N 15 117.193 1 463 569 Q H H 1 7.605 1 464 569 Q N N 15 120.146 1 465 570 E H H 1 8.815 1 466 570 E N N 15 125.031 1 467 571 S H H 1 7.999 1 468 571 S N N 15 113.963 1 469 572 I H H 1 6.963 1 470 572 I N N 15 120.839 1 471 573 D H H 1 8.221 1 472 573 D N N 15 120.607 1 473 574 K H H 1 8.471 1 474 574 K N N 15 110.577 1 475 575 K H H 1 7.695 1 476 575 K N N 15 115.597 1 477 580 D H H 1 7.94 1 478 580 D N N 15 129.176 1 479 581 T H H 1 7.92 1 480 581 T N N 15 115.295 1 481 582 D H H 1 8.477 1 482 582 D N N 15 125.759 1 483 584 W HE1 H 1 10.14 1 484 584 W NE1 H 1 131.131 1 485 585 Q H H 1 8.228 1 486 585 Q N N 15 121.5 1 487 586 L CD1 C 13 24.774 1 488 586 L H H 1 8.179 1 489 586 L N N 15 123.493 1 492 586 L HD1 H 1 0.902 1 493 587 F H H 1 8.927 1 494 587 F N N 15 120.564 1 495 588 S H H 1 8.491 1 496 588 S N N 15 116.853 1 497 589 K H H 1 8.272 1 498 589 K N N 15 125.148 1 499 590 K H H 1 9.035 1 500 590 K N N 15 125.714 1 501 591 S H H 1 8.628 1 502 591 S N N 15 117.949 1 503 592 Q H H 1 7.72 1 504 592 Q N N 15 115.49 1 505 593 E H H 1 8.054 1 506 593 E N N 15 117.488 1 507 594 I H H 1 7.053 1 508 594 I N N 15 112.806 1 509 596 Q H H 1 8.456 1 510 596 Q N N 15 120.73 1 511 597 Q H H 1 8.576 1 512 597 Q N N 15 119.759 1 513 598 M H H 1 9.401 1 514 598 M N N 15 120.151 1 515 599 D H H 1 7.357 1 516 599 D N N 15 116.972 1 517 602 D H H 1 7.383 1 518 602 D N N 15 119.804 1 519 603 S H H 1 8.061 1 520 603 S N N 15 121.228 1 521 604 G H H 1 8.814 1 522 604 G N N 15 109.019 1 523 605 M H H 1 6.87 1 524 605 M N N 15 119.181 1 525 606 F H H 1 8.087 1 526 606 F N N 15 120.065 1 527 607 A H H 1 7.948 1 528 607 A N N 15 119.608 1 529 608 C H H 1 7.095 1 530 608 C N N 15 112.346 1 531 609 K H H 1 7.948 1 532 609 K N N 15 118.448 1 533 610 Y H H 1 9.542 1 534 610 Y N N 15 122.405 1 535 611 A H H 1 7.333 1 536 611 A N N 15 119.249 1 537 612 D H H 1 8.263 1 538 612 D N N 15 120.131 1 539 613 C H H 1 7.203 1 540 613 C N N 15 115.162 1 541 614 I H H 1 8.484 1 542 614 I N N 15 120.137 1 543 615 T H H 1 8.165 1 544 615 T N N 15 109.158 1 545 616 K H H 1 7.126 1 546 616 K N N 15 119.661 1 547 617 D H H 1 7.896 1 548 617 D N N 15 118.551 1 549 618 R H H 1 7.855 1 550 618 R N N 15 117.684 1 551 620 I H H 1 8.365 1 552 620 I N N 15 122.909 1 553 621 D H H 1 7.872 1 554 621 D N N 15 127.17 1 555 622 F H H 1 6.479 1 556 622 F N N 15 114.836 1 557 623 T H H 1 10.893 1 558 623 T N N 15 113.615 1 559 624 Q H H 1 9.761 1 560 624 Q N N 15 119.808 1 561 625 Q H H 1 8.292 1 562 625 Q N N 15 119.007 1 563 626 H H H 1 7.627 1 564 626 H N N 15 117.04 1 565 627 M H H 1 7.554 1 566 627 M N N 15 118.727 1 567 629 Y H H 1 7.389 1 568 629 Y N N 15 120.089 1 569 630 F H H 1 8.629 1 570 630 F N N 15 119.732 1 571 631 R H H 1 8.753 1 572 631 R N N 15 122.645 1 573 632 K H H 1 6.955 1 574 632 K N N 15 116.472 1 575 633 R H H 1 8.412 1 576 633 R N N 15 117.619 1 577 634 M H H 1 8.154 1 578 634 M N N 15 117.932 1 579 635 V CG1 C 13 22.402 1 580 635 V CG2 C 13 24.472 1 581 635 V H H 1 7.322 1 582 635 V N N 15 118.68 1 585 635 V HG1 H 1 0.533 1 588 635 V HG2 H 1 1.092 1 589 636 W H H 1 6.859 1 590 636 W N N 15 118.812 1 591 636 W HE1 H 1 10.124 1 592 636 W NE1 H 1 132.344 1 593 637 E H H 1 8.839 1 594 637 E N N 15 119.355 1 595 638 I H H 1 8.41 1 596 638 I N N 15 119.139 1 597 639 L CD1 C 13 25.313 1 598 639 L CD2 C 13 22.916 1 599 639 L H H 1 7.977 1 600 639 L N N 15 120.642 1 603 639 L HD1 H 1 0.596 1 606 639 L HD2 H 1 0.514 1 607 640 H H H 1 7.669 1 608 640 H N N 15 113.238 1 609 641 R H H 1 7.794 1 610 641 R N N 15 123.547 1 611 642 K H H 1 8.152 1 612 642 K N N 15 122.071 1 613 643 L CD1 C 13 27.478 1 614 643 L CD2 C 13 24.632 1 615 643 L H H 1 8.058 1 616 643 L N N 15 123.331 1 619 643 L HD1 H 1 0.412 1 622 643 L HD2 H 1 0.573 1 623 644 L H H 1 8.748 1 624 644 L N N 15 131.146 1 *referenced using DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) as the H-1 standard with IUPAC-IUB recommended chemical shift referencing ratios. See, Wishart, et al., “1H, 13C and 15N Chemical Shift Referencing in Biomolecular NMR,” J. Biomol. NMR 6: 135-140 (1995); and Markley et al., “Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids,”. Pure & Appl. Chem. 70: 117-142 (1998).

Sequence Listing Isoform 1 SENP1 SEQ ID NO: 1 MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR QGHLDRSFTC STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ KSRNSRSLYL ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS PKKTQRRHVS TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL KDSLFKNGNS CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH HPHHHHSVPH QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD SRARERLRQI EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG HKLTDSEDEF PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI INFYMNMLME RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL GVHWCLAVVD FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK SQEIPQQMNG SDCGMFACKY ADCITKDRPI NFTQQHMPYF RKRMVWEILH RKLL Isoform 2 SENP1 SEQ ID NO: 2 MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR QGHLDRSFTC STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ KSRNSRSLYL ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS PKKTQRRHVS TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL KDSLFKNGNS CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH HPHHHHSVPH QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD SRARERLRQI EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG HKLTDSEDEF PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI INFYMNMLME RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL GVHWCLAVVD FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK SQIPQQMNGS DCGMFACKYA DCITKDRPIN FTQQHMPYFR KRMVWEILHR KLL (Isoform 1) C-Terminal Region SENP1 SEQ ID NO: 3 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK QESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDCGMFACKYADCITKDR PINFTQQHMPYFRKRMVWEILHRKLL (Isoform 1) C-Terminal Region SENP1 C6035 SEQ ID NO: 4 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK QESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDSGMFACKYADCITKDR PINFTQQHMPYFRKRMVWEILHRKLL (Isoform 2) C-Terminal Region SENP1 SEQ ID NO: 5 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK QESIDKKRKEFDTNGWQLFSKKSQIPQQMNGSDCGMFACKYADCITKDRP INFTQQHMPYFRKRMVWEILHRKLL (Isoform 1) Protease Region 450-613 SENP1 SEQ ID NO: 6 LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFT KLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYY DSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMN GSDCGMFACKYADC (Isoform 1) Protease Region 450-613 SENP1 C6035 SEQ ID NO: 7 LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFT KLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYY DSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMN GSDSGMFACKYADC SUMO1 SEQ ID NO: 8 MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKES YCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHST V SUMO1 (1-92) SEQ ID NO: 9 MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKES YCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQ

Embodiments Embodiment 1

A method of detecting binding of an SENP1 polypeptide to a compound, the method comprising:

(i) contacting an SENP1 polypeptide with a compound;

(ii) allowing the compound to bind to the SENP1 polypeptide, thereby forming a SENP1-compound complex;

(iii) detecting the SENP1-compound complex using nuclear magnetic resonance, thereby detecting binding of the SENP1 polypeptide to the compound.

Embodiment 2

The method of embodiment 1, wherein the detecting comprises determining a chemical shift for an amino acid in an active site of the SENP1 polypeptide.

Embodiment 3

The method of embodiment 2, wherein the chemical shift in the presence of the compound is changed relative to the corresponding chemical shift in the absence of the compound.

Embodiment 4

The method of embodiment 2 or 3, wherein the amino acid is an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.

Embodiment 5

The method of embodiment 2 or 3, wherein the amino acid is selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596.

Embodiment 6

The method of embodiment 2 or 3, wherein the amino acid is 5603.

Embodiment 7

The method of embodiment 2 or 3, wherein the amino acid is amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 8

The method of embodiment 1, wherein the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

Embodiment 9

The method of embodiment 1, wherein the SENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1.

Embodiment 10

The method of embodiment 9, wherein the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1.

Embodiment 11

The method of embodiment 10, wherein the mutation is C603S.

Embodiment 12

The method of embodiment 1, wherein the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 13

The method of any one of embodiments 1-12, wherein the SENP1 or SENP1-compound complex is bound to a SUMO protein thereby forming a SENP1-SUMO complex or SENP1-SUMO-compound complex.

Embodiment 14

The method of embodiment 13, wherein the SUMO protein is a truncated SUMO protein.

Embodiment 15

The method of embodiment 2, wherein the active site is a catalytically active site.

Embodiment 16

The method of embodiment 2, wherein the active site is a site that binds to the SUMO protein.

Embodiment 17

The method of any one of embodiments 1-16, wherein the compound is a small molecule.

Embodiment 18

The method of any one of embodiments 1 or 8-17, wherein the detecting comprises producing an NMR spectra of the SENP1-compound complex and identifying a change in the NMR spectra relative to the absence of the compound.

Embodiment 19

The method of embodiment 18, wherein the change is a change in the chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.

Embodiment 20

The method of embodiment 18, wherein the change is a change in the chemical shift of an amino acid selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596.

Embodiment 21

The method of embodiment 18, wherein the change is a change in the chemical shift of the amino acid 5603.

Embodiment 22

The method of embodiment 18, wherein the change is a change in the chemical shift of an amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 23

An aqueous composition comprising an SENP1 polypeptide at a pH from about 6.0 to about 7.5.

Embodiment 24

The aqueous composition of embodiment 23, wherein the pH is about 6.8.

Embodiment 25

The aqueous composition of embodiment 23 or 24, further comprising a buffering agent, reducing agent, solvent, a base, or combinations thereof.

Embodiment 26

The aqueous composition of any one of embodiments 23-25, further comprising sodium phosphate, dimethyl sulfoxide, D2O, sodium azide, dithiothreitol or combinations thereof.

Embodiment 27

The aqueous composition of embodiment 26, wherein the sodium phosphate is present at about 20 mM.

Embodiment 28

The aqueous composition of any one of embodiments 23-27, wherein the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or 7.

Embodiment 29

The aqueous composition of any one of embodiments 23-27, wherein the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 numbered relative to SEQ ID NO:1.

Embodiment 30

The aqueous composition of any one of embodiments 23-29, wherein the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex.

Embodiment 31

The aqueous composition of any one of embodiments 23-29, wherein the SENP1 polypeptide is bound to a compound thereby forming a SENP1-compound complex.

Embodiment 32

The aqueous composition of embodiment 31, wherein the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO-compound complex.

Embodiment 33

The aqueous composition of embodiment 30 or 32, wherein the SUMO protein is a truncated SUMO protein.

Embodiment 34

An NMR apparatus comprising an NMR sample container for NMR analysis, the NMR sample container comprising the aqueous composition of any one of embodiments 23-33.

Embodiment 35

A method of screening for an inhibitor of SENP1 comprising contacting a composition comprising an SENP1 polypeptide with a test compound and detecting whether the test compound binds the SENP1 polypeptide or fragment thereof by nuclear magnetic resonance.

Embodiment 36

The method of embodiment 35, wherein the detecting comprises determining a chemical shift for an amino acid in an active site of the SENP1 polypeptide.

Embodiment 37

The method of embodiment 36, wherein the amino acid is an amino acid of SEQ ID NOs: 3, 4, 5, 6 OR 7.

Embodiment 38

The method of embodiment 36, wherein the amino acid is selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596.

Embodiment 39

The method of embodiment 36, wherein the amino acid is S603.

Embodiment 40

The method of embodiment 36, wherein the amino acid is amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 41

The method of embodiment 35, wherein the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

Embodiment 42

The method of embodiment 35, wherein the SENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1.

Embodiment 43

The method of embodiment 42, wherein the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1.

Embodiment 44

The method of embodiment 43, wherein the mutation is C603S.

Embodiment 45

The method of embodiment 35, wherein the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 46

The method of any one of embodiments 35-45, wherein the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex.

Embodiment 47

The method of embodiment 46, wherein the SUMO protein is a truncated SUMO protein.

Embodiment 48

The method of any one of embodiments 35-47, wherein the chemical shift in the presence of the test compound is changed relative to the corresponding chemical shift in the absence of the test compound.

Embodiment 49

The method of any one of embodiments 35-47, wherein the SENP1 binds the compound forming an SENP1-compound complex and the detecting comprises producing an NMR spectra of the SENP1-compound complex and identifying a change in the NMR spectra relative to the absence of the compound.

Embodiment 50

The method of embodiment 49, wherein the change is a change in the chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.

Embodiment 51

The method of embodiment 49, wherein the change is a change in the chemical shift of an amino acid selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596.

Embodiment 52

The method of embodiment 49, wherein the change is a change in the chemical shift of the amino acid 5603.

Embodiment 53

The method of embodiment 49, wherein the change is a change in the chemical shift of an amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 54

The method of embodiment 49, wherein the change is a change in the chemical shift of an amino acid in the active site of SENP1.

Embodiment 55

The method of embodiment 54, wherein the active site is a catalytically active site.

Embodiment 56

The method of embodiment 54, wherein the active site is a site that binds to the SUMO protein.

Embodiment 57

The method of any one of embodiments 35-56, wherein the test compound is a small molecule.

Embodiment 58

The method of any one of embodiments 35-57, wherein the composition is an aqueous solution.

Embodiment 59

The method of any one of embodiments 35-58, wherein the composition is at a pH from about 6.0 to about 7.5.

Embodiment 60

The method of embodiment 59, wherein the pH is about 6.8.

Embodiment 61

The method of any one of embodiments 35-60, wherein the composition further comprises a buffering agent, solvent, reducing agent, a base, or combinations thereof.

Embodiment 62

The method of any one of embodiments 35-60, further comprising sodium phosphate, D2O, sodium azide, dimethyl sulfoxide, dithiothreitol or combinations thereof.

Embodiment 63

The method of embodiment 62, wherein the sodium phosphate is present at about 20 mM.

Embodiment 64

A method of identifying an SENP1 inhibitor, the method comprising:

combining an SENP1 polypeptide, a SUMO protein, and a test compound in a reaction vessel;

allowing the SENP1 polypeptide, SUMO protein and test compound to form a SENP1-SUMO-compound complex; and detecting the SENP1-SUMO-compound complex thereby identifying the compound as a SENP1 inhibitor.

Embodiment 65

The method of embodiment 64, wherein one or more of the SENP1 polypeptide, SUMO protein or test compound is labeled.

Embodiment 66

The method of embodiment 65, wherein the label is a fluorescent label.

Embodiment 67

The method of any one of embodiments 64-66, wherein the test compound comprises a fluorescent label.

Embodiment 68

The method of any one of embodiments 64-67, wherein binding is detected by fluorescent polarization.

Embodiment 69

The method of embodiment 64, wherein binding is detected by detecting a change in the thermal properties of SENP1.

Embodiment 70

The method of embodiment 69, wherein the thermal property is the melting temperature of SENP1.

Embodiment 71

The method of any one of embodiments 64-70, wherein the SUMO is a truncated SUMO protein.

Embodiment 72

The method of any one of embodiments 64-70, wherein the SUMO comprises amino acid residues 1-92 of the SUMO protein.

Embodiment 73

The method of any one of embodiments 64-70, wherein the SUMO protein comprises SEQ ID NO:8.

Embodiment 74

The method of any one of embodiments 64-70, wherein the SUMO protein comprises SEQ ID NO:9.

Embodiment 75

The method of any one of embodiments 64-74, wherein the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

Embodiment 76

The method of any one of embodiments 64-74, wherein the SENP1 polypeptide comprises amino acid residue 603 of SEQ ID NO:1.

Embodiment 77

The method of any one of embodiments 64-74, wherein the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1.

Embodiment 78

The method of embodiment 77, wherein the mutation is C603S.

Embodiment 79

The method of any one of embodiments 64-74, wherein the SENP1 polypeptide comprises amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 80

The method of any one of embodiments 64 or 71-79,

wherein the detecting is performed using nuclear magnetic resonance.

Embodiment 81

The method of embodiment 80, wherein the detecting comprises producing an NMR spectra of the SENP1-SUMO-compound complex and identifying a change in the NMR spectra relative to the absence of the test compound.

Embodiment 82

The method of embodiment 81, wherein the change is a change in the chemical shift of an amino acid in an active site of the SENP1 polypeptide.

Embodiment 83

The method of embodiment 82, wherein the active site is a catalytically active site.

Embodiment 84

The method of embodiment 82, wherein the active site is a site that binds to the SUMO protein.

Embodiment 85

The method of embodiment 82, wherein the amino acid is an amino acid of SEQ ID NOs: 3, 4, 5, 6 OR 7.

Embodiment 86

The method of embodiment 82, wherein the amino acid is selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596.

Embodiment 87

The method of embodiment 82, wherein the amino acid is S603.

Embodiment 88

The method of embodiment 82, wherein the amino acid is amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.

Embodiment 89

The method of any one of embodiments 64-88, wherein the test compound is a small molecule 

What is claimed:
 1. A method of detecting binding of an SENP1 polypeptide to a compound, the method comprising: (i) contacting an SENP1 polypeptide with a compound; (ii) allowing the compound to bind to the SENP1 polypeptide, thereby forming a SENP1-compound complex; (iii) detecting the SENP1-compound complex using nuclear magnetic resonance, thereby detecting binding of the SENP1 polypeptide to the compound.
 2. The method of claim 1, wherein the detecting comprises determining a chemical shift for an amino acid in an active site of the SENP1 polypeptide.
 3. The method of claim 2, wherein the chemical shift in the presence of the compound is changed relative to the corresponding chemical shift in the absence of the compound.
 4. The method of claim 3, wherein the amino acid is selected from the group consisting of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1.
 5. The method of claim 1, wherein the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or
 7. 6. The method of claim 1, wherein the SENP1 polypeptide comprises a mutation at amino acid residue 603 of SEQ ID NO:1.
 7. The method of claim 1, wherein the SENP1 or SENP1-compound complex is bound to a SUMO protein thereby forming a SENP1-SUMO complex or SENP1-SUMO-compound complex.
 8. The method of claim 1, wherein the detecting comprises producing an NMR spectra of the SENP1-compound complex and identifying a change in the NMR spectra relative to the absence of the compound.
 9. The method of claim 8, wherein the change is a change in the chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or
 7. 10. The method of claim 8, wherein the change is a change in the chemical shift of the amino acid
 5603. 11. The method of claim 8, wherein the change is a change in the chemical shift of an amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.
 12. An aqueous composition comprising an SENP1 polypeptide at a pH from about 6.0 to about 7.5.
 13. The aqueous composition of claim 12, further comprising sodium phosphate, dimethyl sulfoxide, D₂O, sodium azide, dithiothreitol or combinations thereof.
 14. The aqueous composition of claim 13, wherein the sodium phosphate is present at about 20 mM.
 15. The aqueous composition of claim 14, wherein the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or
 7. 16. The aqueous composition of claim 15, wherein the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO complex.
 17. The aqueous composition of claim 15, wherein the SENP1 polypeptide is bound to a compound thereby forming a SENP1-compound complex.
 18. The aqueous composition of claim 17, wherein the SENP1 polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO-compound complex.
 19. A method of screening for an inhibitor of SENP1 comprising contacting a composition comprising an SENP1 polypeptide with a test compound and detecting whether the test compound binds the SENP1 polypeptide or fragment thereof by nuclear magnetic resonance.
 20. A method of identifying an SENP1 inhibitor, the method comprising: combining an SENP1 polypeptide, a SUMO protein, and a test compound in a reaction vessel; allowing the SENP1 polypeptide, SUMO protein and test compound to form a SENP1-SUMO-compound complex; and detecting the SENP1-SUMO-compound complex thereby identifying the compound as a SENP1 inhibitor. 